Htt repressors and uses thereof

ABSTRACT

Disclosed herein are Htt repressors and methods and compositions for use of these Htt repressors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 62/222,588, filed Sep. 23, 2015, the disclosure of whichis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure is in the field of diagnostics and therapeuticsfor Huntington's Disease.

BACKGROUND

Huntington's Disease (HD), also known as Huntington's Chorea, is aprogressive disorder of motor, cognitive and psychiatric disturbances.The mean age of onset for this disease is age 35-44 years, although inabout 10% of cases, onset occurs prior to age 21, and the averagelifespan post-diagnosis of the disease is 15-18 years. Prevalence isabout 3 to 7 among 100,000 people of western European descent.

Huntington's Disease is an example of a trinucleotide repeat expansiondisorders were first characterized in the early 1990s (see Di Prosperoand Fischbeck (2005) Nature Reviews Genetics 6:756-765). These disordersinvolve the localized expansion of unstable repeats of sets of threenucleotides and can result in loss of function of the gene in which theexpanded repeat resides, a gain of toxic function, or both.Trinucleotide repeats can be located in any part of the gene, includingnon-coding and coding gene regions. Repeats located within the codingregions typically involve either a repeated glutamine encoding triplet(CAG) or an alanine encoding triplet (CGA). Expanded repeat regionswithin non-coding sequences can lead to aberrant expression of the genewhile expanded repeats within coding regions (also known as codonreiteration disorders) may cause mis-folding and protein aggregation.The exact cause of the pathophysiology associated with the aberrantproteins is often not known. Typically, in the wild-type genes that aresubject to trinucleotide expansion, these regions contain a variablenumber of repeat sequences in the normal population, but in theafflicted populations, the number of repeats can increase from adoubling to a log order increase in the number of repeats. In HD,repeats are inserted within the N terminal coding region of the largecytosolic protein Huntingtin (Htt). Normal Htt alleles contain 15-20 CAGrepeats, while alleles containing 35 or more repeats can be consideredpotentially HD causing alleles and confer risk for developing thedisease. Alleles containing 36-39 repeats are considered incompletelypenetrant, and those individuals harboring those alleles may or may notdevelop the disease (or may develop symptoms later in life) whilealleles containing 40 repeats or more are considered completelypenetrant. In fact, no asymptomatic persons containing HD alleles withthis many repeats have been reported. Those individuals with juvenileonset HD (<21 years of age) are often found to have 60 or more CAGrepeats. In addition to an increase in CAG repeats, it has also beenshown that HD can involve +1 and +2 frameshifts within the repeatsequences such that the region will encode a poly-serine polypeptide(encoded by AGC repeats in the case of a +1 frameshift) track ratherthan poly-glutamine (Davies and Rubinsztein (2006) Journal of MedicalGenetics 43: 893-896).

In HD, the mutant Htt allele is usually inherited from one parent as adominant trait. Any child born of a HD patient has a 50% chance ofdeveloping the disease if the other parent was not afflicted with thedisorder. In some cases, a parent may have an intermediate HD allele andbe asymptomatic while, due to repeat expansion, the child manifests thedisease. In addition, the HD allele can also display a phenomenon knownas anticipation wherein increasing severity or decreasing age of onsetis observed over several generations due to the unstable nature of therepeat region during spermatogenesis.

Furthermore, trinucleotide expansion in Htt leads to neuronal loss inthe medium spiny gamma-aminobutyric acid (GABA) projection neurons inthe striatum, with neuronal loss also occurring in the neocortex. Mediumspiny neurons that contain enkephalin and that project to the externalglobus pallidum are more involved than neurons that contain substance Pand project to the internal globus pallidum. Other brain areas greatlyaffected in people with Huntington's disease include the substantianigra, cortical layers 3, 5, and 6, the CA1 region of the hippocampus,the angular gyms in the parietal lobe, Purkinje cells of the cerebellum,lateral tuberal nuclei of the hypothalamus, and thecentromedialparafascicular complex of the thalamus (Walker (2007) Lancet369:218-228).

The role of the normal Htt protein is poorly understood, but it may beinvolved in neurogenesis, apoptotic cell death, and vesicle trafficking.In addition, there is evidence that wild-type Htt stimulates theproduction of brain-derived neurotrophic factor (BDNF), a pro-survivalfactor for the striatal neurons. It has been shown that progression ofHD correlates with a decrease in BDNF expression in mouse models of HD(Zuccato et at (2005) Pharmacological Research 52(2): 133-139), and thatdelivery of either BDNF or glial cell line-derived neurotrophic factor(GDNF) via adeno-associated viral (AAV) vector-mediated gene deliverymay protect straital neurons in mouse models of HD (Kells et al, (2004)Molecular Therapy 9(5): 682-688).

Diagnostic and treatment options for HD are currently very limited. Interms of diagnostics, altered (mutant) Htt (mHTT) levels aresignificantly associated with disease burden score, and soluble mHTTspecies increase in concentration with disease progression. However,low-abundance mHTT is difficult to quantify in the patient CNS, whichlimits both study of the role in the neuropathobiology of HD in vivo,and precludes the demonstration of target engagement by HTT-loweringdrugs. See, e.g., Wild et al. (2014) J Neurol Neurosurg Psychiatry85:e4.

With regard to treatment, some potential methodologies designed toprevent the toxicities associated with protein aggregation that occursthrough the extended poly-glutamine tract such as overexpression ofchaperonins or induction of the heat shock response with the compoundgeldanamycin have shown a reduction in these toxicities in in vitromodels. Other treatments target the role of apoptosis in the clinicalmanifestations of the disease. For example, slowing of disease symptomshas been shown via blockage of caspase activity in animal models in theoffspring of a pairing of mice where one parent contained a HD alleleand the other parent had a dominant negative allele for caspase 1.Additionally, cleavage of mutant HD Htt by caspase may play a role inthe pathogenicity of the disease. Transgenic mice carrying caspase-6resistant mutant Htt were found to maintain normal neuronal function anddid not develop striatal neurodegeneration as compared to mice carryinga non-caspase resistant mutant Htt allele (see Graham et at (2006) Cell125: 1179-1191). Molecules which target members of the apoptotic pathwayhave also been shown to have a slowing effect on symptomology. Forexample, the compounds zVAD-fmk and minocycline, both of which inhibitcaspase activity, have been shown to slow disease manifestation in mice.The drug remacemide has also been used in small HD human trials becausethe compound was thought to prevent the binding of the mutant Htt to theNDMA receptor to prevent the exertion of toxic effects on the nervecell. However, no statistically significant improvements were observedin neuron function in these trials. In addition, the Huntington StudyGroup conducted a randomized, double-blind study using Co-enzyme Q.Although a trend towards slower disease progression among patients thatwere treated with coenzyme Q10 was observed, there was no significantchange in the rate of decline of total functional capacity. (Di Prosperoand Fischbeck, ibid).

Recombinant transcription factors and nucleases comprising the DNAbinding domains from zinc finger proteins (“ZFPs”), TAL-effector domains(“TALEs”) and CRISPR/Cas transcription factor systems (including Casand/or Cfp1 systems) have the ability to regulate gene expression ofendogenous genes. See, e.g., U.S. Pat. Nos. 9,045,763; 9,005,973;8,956,828; 8,945,868; 8,586,526; 6,534,261; 6,599,692; 6,503,717;6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796;7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410;20050208489; 20050026157; 20050064474; 20060063231; 20080159996;201000218264; 20120017290; 20110265198; 20130137104; 20130122591;20130177983 and 20130177960 and 20150056705 and U.S. application Ser.No. 14/706,747; Perez-Pinera et al. (2013) Nature Methods 10:973-976;Platek et al. (2014) Plant Biotechnology J. doi: 10.1111/pbi.12284), thedisclosures of which are incorporated by reference in their entiretiesfor all purposes. Further, targeted nucleases are being developed basedon the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’,see Swarts et at (2014) Nature 507(7491): 258-261), which also may havethe potential for uses in genome editing and gene therapy. Clinicaltrials using these engineered transcription factors containing zincfinger proteins have shown that these novel transcription factors arecapable of treating various conditions. (see, e.g., Yu et al. (2006)FASEB J. 20:479-481). Nuclease-mediated cleavage involves the use ofengineered nucleases to induce a double strand break (DSB) or a nick ina target DNA sequence such that repair of the break by an error bornprocess such as non-homologous end joining (NHEJ) or repair using arepair template (homology directed repair or HDR) can result in theknock out of a gene or the insertion of a sequence of interest (targetedintegration). Introduction of a double strand break in the absence of anexternally supplied repair template (e.g. “donor” or “transgene”) iscommonly used for the inactivation of the targeted gene via mutations(insertions and/or deletions known as “indels”) introduced by thecellular NHEJ pathway. For instance, U.S. Patent Publication 20110082093discloses specific zinc finger proteins targeted to Htt and U.S. PatentPublication No. 20130253040 relates to DNA-binding proteins thatmodulate expression of an HD allele such as Htt. U.S. Publication No.20150335708 relates to methods of modifying medium spiny neurons.

However, there remains a need for methods for the diagnosis, study,treatment and/or prevention of Huntington's Disease, including formodalities that exhibit widespread delivery to the brain.

SUMMARY

Disclosed herein are methods and compositions for diagnosing, preventingand/or treating Huntington's Disease. In particular, provided herein aremethods and compositions for modifying (e.g., modulating expression of)an HD Htt allele so as to treat Huntington Disease, including Httrepressors (that repress Htt expression). The compositions (Httrepressors) described herein provide a therapeutic benefit in subjects,for example by reducing cell death, decreasing apoptosis, increasingcellular function (metabolism) and/or reducing motor deficiency in thesubjects. Also provided are methods and compositions that allow forbi-directional axonal transport in a primate brain. Surprisingly andunexpectedly, the present inventors have found that unlike other AAVserotypes that have been used, AAV9 exhibits widespread deliverythroughout the brain, including anterograde and retrograde axonaltransport to brain regions distant from the site of AAV9 administration.Thus, described herein is a non-naturally occurring zinc finger proteinthat binds to an Htt gene, the zinc finger protein comprising 5 zincfinger domains ordered F1 to F5, wherein the zinc finger domainscomprise the recognition helix regions sequences shown in a single rowof Table 1.

Thus, in one aspect, engineered (non-naturally occurring) Htt repressorsare provided. The repressors may comprise systems (e.g., zinc fingerproteins, TAL effector (TALE) proteins or CRISPR/dCas-TF) that modulateexpression of a HD allele (e.g., Htt). Engineered zinc finger proteinsor TALEs are non-naturally occurring zinc finger or TALE proteins whoseDNA binding domains (e.g., recognition helices or RVDs) have beenaltered (e.g., by selection and/or rational design) to bind to apre-selected target site. Any of the zinc finger proteins describedherein may include 1, 2, 3, 4, 5, 6 or more zinc fingers, each zincfinger having a recognition helix that binds to a target subsite in theselected sequence(s) (e.g., gene(s)). Similarly, any of the TALEproteins described herein may include any number of TALE RVDs. In someembodiments, at least one RVD has non-specific DNA binding. In someembodiments, at least one recognition helix (or RVD) is non-naturallyoccurring. In certain embodiments, the zinc finger proteins have therecognition helices in the proteins designated 45643 or 46025 (Table 1).In certain embodiments, the repressor comprises a DNA-binding domain(ZFP, TALE, single guide RNA) operably linked to a transcriptionalrepression domain. In some embodiments these ZFP-TFs, CRISPR/dCas-TFs orTALE-TFs include protein interaction domains (or “dimerization domains”)that allow multimerization when bound to DNA.

In certain embodiments, the zinc finger proteins (ZFPs), Cas protein ofa CRISPR/Cas system or TALE proteins as described herein can be placedin operative linkage with a regulatory domain (or functional domain) aspart of a fusion protein. The functional domain can be, for example, atranscriptional activation domain, a transcriptional repression domainand/or a nuclease (cleavage) domain. By selecting either an activationdomain or repression domain for use with the DNA-binding domain, suchmolecules can be used either to activate or to repress gene expression.In some embodiments, a molecule comprising a ZFP, dCas or TALE targetedto a mutant Htt as described herein fused to a transcriptionalrepression domain that can be used to down-regulate mutant Httexpression is provided. In some embodiments, a fusion protein comprisinga ZFP, CRISPR/Cas or TALE targeted to a wild-type Htt allele fused to atranscription activation domain that can up-regulate the wild type Httallele is provided. In certain embodiments, the activity of theregulatory domain is regulated by an exogenous small molecule or ligandsuch that interaction with the cell's transcription machinery will nottake place in the absence of the exogenous ligand, while in otherembodiments, the exogenous small molecule or ligand prevents theinteraction. Such external ligands control the degree of interaction ofthe ZFP-TF, CRISPR/Cas-TF or TALE-TF with the transcription machinery.The regulatory domain(s) may be operatively linked to any portion(s) ofone or more of the ZFPs. dCas or TALEs, including between one or moreZFPs, dCas or TALEs, exterior to one or more ZFPs, dCas or TALEs and anycombination thereof. Any of the fusion proteins described herein may beformulated into a pharmaceutical composition.

In some embodiments, the engineered DNA binding domains as describedherein can be placed in operative linkage with nuclease (cleavage)domains as part of a fusion protein. In some embodiments, the nucleasecomprises a Ttago nuclease. In other embodiments, nuclease systems suchas the CRISPR/Cas system may be utilized with a specific single guideRNA to target the nuclease to a target location in the DNA. In certainembodiments, such nucleases and nuclease fusions may be utilized fortargeting mutant Htt alleles in stem cells such as induced pluripotentstem cells (iPSC), human embryonic stem cells (hESC), mesenchymal stemcells (MSC) or neuronal stem cells wherein the activity of the nucleasefusion will result in an Htt allele containing a wild type number of CAGrepeats. Thus, any of the Htt repressors described herein can furthercomprise a dimerization domain and/or a functional domain (e.g.,transcriptional activation domain, a transcriptional repression domainor a nuclease domain). In certain embodiments, pharmaceuticalcompositions comprising the modified cells (e.g., stem cells) areprovided.

In yet another aspect, a polynucleotide encoding one or more of the DNAbinding proteins described herein is provided. In certain embodiments,the polynucleotide is carried on a viral (e.g., AAV or Ad) vector and/ora non-viral (e.g., plasmid or mRNA vector). Host cells comprising thesepolynucleotides (e.g., AAV vectors) and/or pharmaceutical compositionscomprising the polynucleotides, proteins and/or host cells as describedherein are also provided.

In other aspects, the invention comprises delivery of a donor nucleicacid to a target cell. The donor may be delivered prior to, after, oralong with the nucleic acid encoding the nuclease(s). The donor nucleicacid may comprise an exogenous sequence (transgene) to be integratedinto the genome of the cell, for example, an endogenous locus. In someembodiments, the donor may comprise a full length gene or fragmentthereof flanked by regions of homology with the targeted cleavage site.In some embodiments, the donor lacks homologous regions and isintegrated into a target locus through homology independent mechanism(i.e. NHEJ). The donor may comprise any nucleic acid sequence, forexample a nucleic acid that, when used as a substrate forhomology-directed repair of the nuclease-induced double-strand break,leads to a donor-specified deletion to be generated at the endogenouschromosomal locus or, alternatively (or in addition to), novel allelicforms of (e.g., point mutations that ablate a transcription factorbinding site) the endogenous locus to be created. In some aspects, thedonor nucleic acid is an oligonucleotide wherein integration leads to agene correction event, or a targeted deletion.

In some embodiments, the polynucleotide encoding the DNA binding proteinis an mRNA. In some aspects, the mRNA may be chemically modified (Seee.g. Kormann et al, (2011) Nature Biotechnology 29(2):154-157). In otheraspects, the mRNA may comprise an ARCA cap (see U.S. Pat. Nos. 7,074,596and 8,153,773). In further embodiments, the mRNA may comprise a mixtureof unmodified and modified nucleotides (see U.S. Patent Publication2012-0195936).

In yet another aspect, a gene delivery vector comprising any of thepolynucleotides described herein is provided. In certain embodiments,the vector is an adenovirus vector (e.g., an Ad5/F35 vector), alentiviral vector (LV) including integration competent orintegration-defective lentiviral vectors, or an adenovirus associatedviral vector (AAV). In certain embodiments, the AAV vector is an AAV6 orAAV9 vector. Thus, also provided herein are adenovirus (Ad) vectors, LVor adenovirus associate viral vectors (AAV) comprising a sequenceencoding at least one nuclease (ZFN or TALEN) and/or a donor sequencefor targeted integration into a target gene. In certain embodiments, theAd vector is a chimeric Ad vector, for example an Ad5/F35 vector. Incertain embodiments, the lentiviral vector is an integrase-defectivelentiviral vector (IDLV) or an integration competent lentiviral vector.In certain embodiments the vector is pseudo-typed with a VSV-G envelope,or with other envelopes.

Additionally, pharmaceutical compositions comprising the nucleic acidsand/or proteins (e.g., ZFPs, Cas or TALEs or fusion proteins comprisingthe ZFPs, Cas or TALEs) are also provided. For example, certaincompositions include a nucleic acid comprising a sequence that encodesone of the ZFPs, Cas or TALEs described herein operably linked to aregulatory sequence, combined with a pharmaceutically acceptable carrieror diluent, wherein the regulatory sequence allows for expression of thenucleic acid in a cell. In certain embodiments, the ZFPs, CRISPR/Cas orTALEs encoded are specific for a HD Htt allele. In some embodiments,pharmaceutical compositions comprise ZFPs, CRISPR/Cas or TALEs thatmodulate a HD Htt allele and ZFPs, CRISPR/Cas or TALEs that modulate aneurotrophic factor. Protein based compositions include one of moreZFPs. CRISPR/Cas or TALEs as disclosed herein and a pharmaceuticallyacceptable carrier or diluent.

In yet another aspect also provided is an isolated cell comprising anyof the proteins, polynucleotides and/or compositions as describedherein.

In another aspect, described herein are methods of modifying expressionof an Htt gene in a cell (e.g., neuronal cell in vitro or in vivo in abrain of a subject, e.g., the striatum), the method comprisingadministering to the cell one or more proteins, polynucleotides and/orcells as described herein. The Htt gene may comprise at least onewild-type and/or mutant Htt allele. In certain embodiments, Httexpression is repressed.

In another aspect, provided herein are methods for treating and/orpreventing Huntington's Disease using the methods and compositions(proteins, polynucleotides and/or cells) described herein. In someembodiments, the methods involve compositions where the polynucleotidesand/or proteins may be delivered using a viral vector, a non-viralvector (e.g., plasmid) and/or combinations thereof. In some embodiments,the methods involve compositions comprising stem cell populationscomprising a ZFP or TALE, or altered with the ZFNs, TALENs, Ttago or theCRISPR/Cas nuclease system of the invention. The subject may comprise atleast one mutant and/or wild-type Htt allele.

In a still further aspect, described here is a method of delivering arepressor of Htt to the brain of the subject using an AAV (e.g., AAV9)vector. Delivery may be to any brain region, for example, the striatum(e.g., putamen) by any suitable means including via the use of acannula. In some embodiments, delivery is through direct injection intothe intrathecal space. In further embodiments, delivery in throughintravenous injection. The AAV9 vector provides widespread delivery ofthe repressor to brain of the subject, including via anterograde andretrograde axonal transport to brain regions not directly administeredthe vector (e.g., delivery to the putamen results in delivery to otherstructures such as the cortex, substantia nigra, thalamus, etc. Incertain embodiments, the subject is a human and in other embodiments,the subject is a non-human primate.

Thus, in other aspects, described herein is a method of preventingand/or treating HD in a subject, the method comprising administering arepressor of a mutant Htt allele to the subject. The repressor may beadministered in polynucleotide form (e.g., using a viral (e.g., AAV)and/or non-viral vector (e.g., plasmid and/or mRNA), in protein formand/or via a pharmaceutical composition as described herein (e.g., apharmaceutical compositions comprising a polynucleotide, AAV vector,protein and/or cell as described herein). In certain embodiments, therepressor is administered to the CNS (e.g., putamen) of the subject. Therepressor may provide therapeutic benefits, including, but not limitedto, reducing the formation of Htt aggregates in HD neurons of a subjectwith HD; reducing cell death in a neuron or population of neurons (e.g.,an HD neuron or population of HD neurons); and/or reducing motordeficits (e.g., clasping) in HD subjects.

In any of the methods described herein, the repressor of the mutant Httallele may be a ZFP-TF, for example a fusion protein comprising a ZFPthat binds specifically to a mutant Htt allele and a transcriptionalrepression domain (e.g., KOX, KRAB, etc.). In other embodiments, therepressor of the mutant Htt allele may be a TALE-TF, for example afusion protein comprising a TALE polypeptide that binds specifically toa mutant Htt allele and a transcriptional repression domain (e.g., KOX,KRAB, etc.). In some embodiments, the mutant Htt allele repressor is aCRISPR/Cas-TF where the nuclease domains in the Cas protein have beeninactivated such that the protein no longer cleaves DNA. The resultantCas RNA-guided DNA binding domain is fused to a transcription repressor(e.g. KOX, KRAB etc.) to repress the mutant Htt allele. In still furtherembodiments, the repressor may comprise a nuclease (e.g., ZFN, TALENand/or CRISPR/Cas system) that represses the mutant Htt allele bycleaving and thereby inactivating the mutant Htt allele. In certainembodiments, the nuclease introduces an insertion and/or deletion(“indel”) via non-homologous end joining (NHEJ) following cleavage bythe nuclease. In other embodiments, the nuclease introduces a donorsequence (by homology or non-homology directed methods), in which thedonor integration inactivates the mutant Htt allele.

In any of the methods described herein, the repressor may be deliveredto the subject (e.g., brain) as a protein, polynucleotide or anycombination of protein and polynucleotide. In certain embodiments, therepressor(s) is(are) delivered using an AAV (e.g., AAV9) vector. Inother embodiments, at least one component of the repressor (e.g., sgRNAof a CRISPR/Cas system) is delivered as in RNA form. In otherembodiments, the repressor(s) is(are) delivered using a combination ofany of the expression constructs described herein, for example onerepressor (or portion thereof) on one expression construct (e.g., AAVsuch as AAV9) and one repressor (or portion thereof) on a separateexpression construct (AAV or other viral or non-viral construct).

Furthermore, in any of the methods described herein, the repressors canbe delivered at any concentration (dose) that provides the desiredeffect. In preferred embodiments, the repressor is delivered using anadeno-associated virus vector at 10,000-500,000 vector genome/cell (orany value therebetween). In certain embodiments, the repressor isdelivered using a lentiviral vector at MOI between 250 and 1,000 (or anyvalue therebetween). In other embodiments, the repressor is deliveredusing a plasmid vector at 150-1,500 ng/100,000 cells (or any valuetherebetween). In other embodiments, the repressor is delivered as mRNAat 150-1,500 ng/100,000 cells (or any value therebetween).

In any of the methods described herein, the method can yield about 70%or greater, about 75% or greater, about 85% or greater, about 90% orgreater, about 92% or greater, or about 95% or greater repression of themutant Htt alleles in one or more HD neurons of the subject.

In further aspects, the invention described herein comprises one or moreHtt-modulating transcription factors, such as a Htt-modulatingtranscription factors comprising one or more of a zinc finger protein(ZFP TFs), a TALEs (TALE-TF), and a CRISPR/Cas-TFs for example, ZFP-TFs,TALE-TFs or CRISPR/Cas-TFs. In certain embodiments, the Htt-modulatingtranscription factor can repress expression of a mutant Htt allele inone or more HD neurons of a subject. The repression can be about 70% orgreater, about 75% or greater, about 85% or greater, about 90% orgreater, about 92% or greater, or about 95% or greater repression of themutant Htt alleles in the one or more HD neurons of the subject ascompared to untreated (wild-type) neurons of the subject. In certainembodiments, the Htt-modulating transcription factor can be used toachieve one or more of the methods described herein.

In some embodiments, therapeutic efficacy is measured using the UnifiedHuntington's Disease Rating Scale (UHDRS) (Huntington Study Group (1996)Mov Disord 11(2):136-142) for analysis of overt clinical symptoms. Inother embodiments, efficacy in patients is measured using PET and Millimaging. In some embodiments, treatment with the mutant Htt modulatingtranscription factor prevents any further development of overt clinicalsymptoms and prevents any further loss of neuron functionality. In otherembodiments, treatment with the mutant Htt modulating transcriptionfactor improves clinical symptoms and improves neuron function.

Also provided is a kit comprising one or more of the AAV9 Htt-modulators(e.g., repressors) and/or polynucleotides comprising components ofand/or encoding the Htt-modulators (or components thereof) as describedherein. The kits may further comprise cells (e.g., neurons), reagents(e.g., for detecting and/or quantifying mHtt protein, for example inCSF) and/or instructions for use, including the methods as describedherein.

Thus, the present disclosure encompasses, but is not limited to, thefollowing numbered embodiments:

1. A non-naturally occurring zinc finger protein that binds to an Httgene, the zinc finger protein comprising 5 zinc finger domains orderedF1 to F5, wherein the zinc finger domains comprise the recognition helixregions sequences shown in a single row of Table 1.

2. The Htt repressor of 1, further comprising a dimerization domain thatallows multimerization of zinc finger proteins when bound to DNA.

3. A fusion protein comprising a zinc finger protein of 1 or 2 and afunctional domain.

4. The fusion protein of 3, wherein the functional domain is selectedfrom the group consisting of a transcriptional activation domain, atranscriptional repression domain, and a nuclease domain.

5. A polynucleotide encoding one or more zinc finger proteins of 1 to 2or one or more fusion proteins of 3 or 4.

6. An AAV vector comprising the polynucleotide of 5.

7. The AAV vector of 6, wherein the vector is an AAV9 vector.

8. A host cell comprising one or more zinc finger proteins of 1 to 2 orone or more fusion proteins of 3 or 4, one or more polynucleotidesaccording to 5 or one or more AAV vectors according to 6 or 7.

9. A pharmaceutical composition comprising one or more zinc fingerproteins of 1 to 2 or one or more fusion proteins of 3 or 4, one or morepolynucleotides according to claim 5 or one or more AAV vectorsaccording to 6 or 7.

10. A method of modifying expression of an Htt gene in a cell, themethod comprising administering to the cell one or more polynucleotidesaccording to 5 or one or more AAV vectors according to 6 or 7.

11. The method of 10, wherein the Htt gene comprises at least one mutantallele.

12. The method of 10, wherein the Htt gene is wild-type.

13. The method of any of 10 to 12, wherein the fusion protein comprisesa repression domain and expression of the Htt gene is repressed.

14. The method of any of 10 to 13, wherein the cell is a neuronal cell.

15. The method of 14 wherein the neuronal cell is in a brain.

16. The method of 15, wherein the neuronal cell is in the striatum ofthe brain.

17. A method of treating and/or preventing Huntington's Disease in asubject in need thereof, the method comprising administering one or morepolynucleotides according to 5 or one or more AAV vectors according to 6or 7 to the subject in need thereof.

18. A method of modifying expression of an Htt gene in a cell, themethod comprising administering to the cell one or more AAV9 vectors,the AAV9 vectors encoding one or more Htt repressors.

19. The method of 18, wherein the Htt gene comprises at least one mutantallele.

20. The method of 18, wherein the Htt gene is wild-type.

21. The method of any of 18 to 21, wherein the fusion protein comprisesa repression domain and expression of the Htt gene is repressed.

22. The method of any of 18 to 21, wherein the cell is a neuronal cell.

23. The method of 22, wherein the neuronal cell is in a brain.

24. The method of 23, wherein the neuronal cell is in the striatum ofthe brain.

25. A method of treating and/or preventing Huntington's Disease in asubject in need thereof, the method comprising administering to thesubject in need thereof one or more AAV9 vectors, the AAV9 vectorsencoding one or more Htt repressors.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1J are images depicting results following infusion ofAAV9 vectors into NHP putamen. Comparative panels for the MRI-guidedinfusion of AAV9-GFP (100 μL) into the bilateral putamen of 2 normalnonhuman primates (designated NHP-1 and NHP-2) are shown in FIGS. 1A and1B and 3 dimensional reconstructs of each subject are shown in FIGS. 1Cand 1D. Reconstructed volumes for each putamen (n=4), labeled in bluemeasured approximately 850 μL for NHP-1 and 625 μL of NHP-2. Theinfusate containing AAV9-GFT particles and chelated gadolinium imagingreagent, labeled in orange, distributed into approximately 350 μL foreach infusion. FIGS. 1E, 1F and 1G show coronal, sagittal left andsagittal right views (respectively) of cannula trajectory paths of NHP-1and FIGS. 1H, 1I and 1J show coronal, sagittal left and sagittal rightviews (respectively) of cannula trajectory paths for NHP-2.

FIGS. 2A through 211 show AAV9-mediated GFP expression in the putamen ofNHPs and innate immune status. FIGS. 2A and 2B show GFP staining withinthe putamen. High magnification images of the boxed areas are shownbelow and depict NeuN-positive cells in putamen administered either ahigh dose (“HD”, left hemisphere 1.5×10¹³ vg/mL) or low dose (“LD”,right hemisphere, 1.5 and 10¹² vg/mL) of vector. FIGS. 2C and 2D aregraphs depicting the primary area of transduction (PAT) shown in whiteand the area within the putamen “outside” the GFP-positive signal is“oPAT” and shown in grey. Counts of NeuN-positive cells in three coronalplants at PAT and oPAT, performed alongside naive control NHPs withinthe putamen showed no detectible difference in NeuN signal when the lowand high dose hemispheres were compared. Counts of co-localizedGFP-positive/NeuN-positive cells revealed transduction is about 74%efficient in PAT with low to null efficiency in oPAT. Data arerepresented as mean number of cells ±SD. P-values (*) of <0.01 fortransduced cells within PAT and oPAT, Wilcoxon sign-ranked S test. Scalebars for FIGS. 2A through 2D: 50 μM. FIGS. 2E, 2F, 2G and 2H show distal(FIGS. 2E and 2F) and proximal (FIGS. 2G and 2H) comparison of innateimmune status. The up-regulation of positively stained MHCII (FIGS. 2Eand 2G, scale bar of 50 μm) and Iba1 activation (FIGS. 2F and 2H, scalebar of 200 μm) is shown proximal to the site of infusion in comparisonto the distal site.

FIGS. 3A through 3F are imaged showing cellular tropism of the AAV9vectors at the site of infusion in the putamen of the indicated targets(NeuN, GFAP, Iba1 in red), GFP (green) and double-labeledimmmunofluorescence (yellow). FIGS. 3A, 3B and 3C show results in NHP-1and FIGS. 3D, 3E and 3F show results in NHP-2. Double-labelimmunofluorescence revealed GFP expression in both NeuN-positive andGFAP-positive cell bodies. In contrast, capsids did not transduceIba1-positive cell bodies, indicating the AAV9 does not appear totransduce microglia. Scale bars: 50 μm.

FIGS. 4A and 4B shows retrograde axonal transport of AAV9 from putamento cortex. Coronal brain sections processed from NHP-1 (FIG. 4A) andNHP-2 (FIG. 4B) anterior (i and ii) and posterior (iii and iv) to thecannula tract showed robust axonal transport of AAV9-GFP alongcortico-striatal projections. High magnification images of the areaswithin the black squared labeled “i” “ii” “iii” and “iv.” show increasedGFP expression in cortical cell bodies and fibers within the hemispherereceiving high-dose vector indicating a dose effect with respect to thecontralateral side. Scale bars: 200 μm.

FIGS. 5A and 5B depict axonal transport to distal regions. FIG. 5A showsresults from NHP-1 and FIG. 5B shows results from NHP-2. The infusion ofAAV9 into the putamen resulted in GFP-positive cells in many distalregions known to receive effects from the putamen. Shown is the anti-GFPimmunohistochemistry with approximate position of each coronal section12 mm posterior to the cannula tract. High magnification images belowcorrespond to left and right hemispheres of the globus pallidus (“GP”),thalamus, subthalamic nuclease (“STN”), medial forebrain bundle (“MFB”)and substantia nigra (“SN”). Scale bars: 500 μM.

FIGS. 6A and 6B depict axonal transport of AAV to substantia nigra parsreticulata and pars compacta. FIG. 6A depicts results in NHP-1 and FIG.6B depicts results in NHP-2. Double-label immunofluorescence (yellow) isvisible for GFP-positive (green) and tyrosine hydroxylase-positive (red)cell codies and fibers of the substantia nigra pars compacta (“Snc”) andin fibers of the parts reticulata (“SNr”). Tyrosine hydroxylase (“TH”)staining identifies dopaminergic neurons. Transduction of cell bodies inthe SNr and SNc demonstrates both anterograde and retrograde axonaltransport respectively. Increased expression of GFP was observed betweenthe ipsilateral (high-dose) and contralateral (low-dose) hemispheres.Scale bars: 500 μm.

FIG. 7 is a schematic depicting neuronal connection pathways in primatebrain. Illustrated anterograde and retrograde transport system and doseeffect between high dose (left) and low dose (right) NHP brainhemispheres. AAV9-GFP viral particles at the striatal injection site(“Str”), shaded in grey, were transported retrogradely to secondarybrain areas including cortex (“Ctx”), thalamus (“Th”) and substantianigra parts compacta (“SNc”). Anterograde transport of viral particlestargeted parts reticulata (“SNr”). Transport either by perivascularspread or anterograde axonal means to the globus pallidus (“GP”) led inturn to axonal transport to the subthalamic nucleus (“STN”). Thepresence of neuronal GFP-positive fibers is indicated by the greystrokes and GFP-positive cells bodies by the black dots. Absence oftransduced cell bodies is indicated by empty circles.

FIG. 8 is a graph showing the expression profiles of either the wildtype Htt gene (grey bars, CAG18) or the mutant Htt gene (black bars,CAG45) in fibroblasts from HD patients that have been treated witheither ZFP 46025, ZFP 45643 or a GFP control. The data demonstrates thatwhile the expression of the wild type Htt allele is fairly constant inall samples, the cells that had been treated with the mutantHtt-specific ZFPs had decreased expression of the mutant Htt at thehigher doses of ZFP mRNA.

FIG. 9 is a graph showing expression profiles of either the wild typeHtt gene (grey bars, CAG21) or the mutant Htt gene (black bars, CAG38)in fibroblasts from HD patients that have been treated with either ZFP46025, ZFP 45643 or a GFP control. The data demonstrates that while theexpression of the wild type Htt allele is fairly constant in allsamples, the cells that had been treated with the mutant Htt-specificZFPs had decreased expression of the mutant Htt at the higher doses ofZFP mRNA.

FIG. 10 is a graph showing expression profiles of either the wild typeHtt gene (grey bars, CAG17) or the mutant Htt gene (black bars, CAG48)in neurons that have been differentiated from HD embryonic stem cells.The data demonstrates that at the higher doses of mRNA encoding theZFPs, the mutant Htt gene was repressed while the wild type Htt allelewas not.

FIGS. 11A and 11B are graphs showing Htt repression in CAG17/48differentiated neurons where the ZFPs using were delivered using eitherAAV6 or AAV9 viral vectors. AAV6 vectors (FIG. 11A) or AAV9 vectors(FIG. 11B) encoding ZFP 46025, 45643 or GFP control were used to infectneurons differentiated from HD embryonic stem cells (ESC) GENEA020(GENEA/CHDI) in duplicate. The data demonstrates that the mutant Httallele was repressed by the ZFPs when delivered either by AAV6 or AAV9as compared to cells treated with the GFP control or those that had beenput through a mock infection protocol.

FIGS. 12A and 12B are graphs depicting the effect of treatment with theZFPs on phenotypic characteristics of the HD neurons. FIG. 12Ademonstrates that ATP levels in the neurons are higher in the HD neuronstreated with the ZFPs than in the cells treated with the VENUS controlor the mock treated cells. In comparison, the wild type neurons do notshow any effect from the ZFP treatment. FIG. 12B shows the percent ofcells that are TUNEL positive (a marker for apoptosis), and demonstratesthat the HD neurons treated with the ZFPs have less numbers of TUNELpositive cells than the control samples. For comparison, the graph alsodepicts the data from wild type neurons.

FIGS. 13A through 13C are graphs depicting the in vivo activity of ZFPswere tested in HdhQ50/Hdh+ (Q50) heterozygous mice by intrastriatalinjection of AAV9 vectors encoding ZFP 46025, ZFP 45643 or GFP control.The Q50 mice contain a knock-in allele where exon 1 of the endogenousmouse Hdh gene was replaced with exon 1 of the human Htt gene with 48CAGs. At 5 weeks after injection, allele-specific qRT-PCR analysis oftreated striatum showed that ZFP 45643 and ZFP 46025 repressed themutant Htt allele (Q50) by 79% and 74%, respectively, relative tovehicle injected control; the wild type allele (Q7) was not regulated byeither ZFP (FIGS. 13A and 13B). Activity of ZFP 45643 was also tested at12 weeks after injection (FIG. 13C), and significant repression (70%) ofmutant Htt (Q50) was observed with no repression of the wild type allele(Q7).

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for widespread CNSdelivery of compositions for detecting, monitoring disease progression,treating and/or preventing Huntington's disease (HD). In particular, thecompositions and methods described herein use AAV9 vectors for deliveryof Htt repressors, which provides for the spread of functional Httrepressors beyond the site of delivery. The Htt repressors (e.g.,Htt-modulating transcription factors, such as Htt-modulatingtranscription factors comprising zinc finger proteins (ZFP TFs), TALEs(TALE-TF), or CRISPR/Cas-TFs for example, ZFP-TFs, TALE-TFs orCRISPR/Cas-TFs which repress expression of a mutant Htt allele) modifythe CNS such that the effects and/or symptoms of HD are reduced oreliminated, for example by reducing the aggregation of Htt in HDneurons, by increasing HD neuron energetics (e.g., increasing ATPlevels), by reducing apoptosis in HD neurons and/or by reducing motordeficits in HD subjects.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acid.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Pat. No. 8,586,526.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g., Swarts et at (2014) Nature 507(7491):258-261, G. Sheng etal., (2013) Proc. Natl. Acad. Sci. U.S.A. 111, 652). A “TtAgo system” isall the components required including, for example, guide DNAs forcleavage by a TtAgo enzyme. “Recombination” refers to a process ofexchange of genetic information between two polynucleotides, includingbut not limited to, donor capture by non-homologous end joining (NHEJ)and homologous recombination. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to resynthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

Zinc finger binding domains or TALE DNA binding domains can be“engineered” to bind to a predetermined nucleotide sequence, for examplevia engineering (altering one or more amino acids) of the recognitionhelix region of a naturally occurring zinc finger protein or byengineering the RVDs of a TALE protein. Therefore, engineered zincfinger proteins or TALEs are proteins that are non-naturally occurring.Non-limiting examples of methods for engineering zinc finger proteins orTALEs are design and selection. A “designed” zinc finger protein or TALEis a protein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. A “selected” zinc finger protein or TALE is aprotein not found in nature whose production results primarily from anempirical process such as phage display, interaction trap or hybridselection. See, for example, U.S. Pat. Nos. 8,586,526; 6,140,081;6,453,242; 6,746,838; 7,241,573; 6,866,997; 7,241,574 and 6,534,261; seealso WO 03/016496.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid. The term also includes systems in which apolynucleotide component associates with a polypeptide component to forma functional molecule (e.g., a CRISPR/Cas system in which a single guideRNA associates with a functional domain to modulate gene expression).

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “multimerization domain”, (also referred to as a “dimerization domain”or “protein interaction domain”) is a domain incorporated at the amino,carboxy or amino and carboxy terminal regions of a ZFP TF or TALE TF.These domains allow for multimerization of multiple ZFP TF or TALE TFunits such that larger tracts of trinucleotide repeat domains becomepreferentially bound by multimerized ZFP TFs or TALE TFs relative toshorter tracts with wild-type numbers of lengths. Examples ofmultimerization domains include leucine zippers. Multimerization domainsmay also be regulated by small molecules wherein the multimerizationdomain assumes a proper conformation to allow for interaction withanother multimerization domain only in the presence of a small moleculeor external ligand. In this way, exogenous ligands can be used toregulate the activity of these domains.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALE protein asdescribed herein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to an activation domain, the ZFP or TALEDNA-binding domain and the activation domain are in operative linkageif, in the fusion polypeptide, the ZFP or TALE DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe activation domain is able to upregulate gene expression. ZFPs fusedto domains capable of regulating gene expression are collectivelyreferred to as “ZFP-TFs” or “zinc finger transcription factors”, whileTALEs fused to domains capable of regulating gene expression arecollectively referred to as “TALE-TFs” or “TALE transcription factors.”When a fusion polypeptide in which a ZFP DNA-binding domain is fused toa cleavage domain (a “ZFN” or “zinc finger nuclease”), the ZFPDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site. When a fusionpolypeptide in which a TALE DNA-binding domain is fused to a cleavagedomain (a “TALEN” or “TALE nuclease”), the TALE DNA-binding domain andthe cleavage domain are in operative linkage if, in the fusionpolypeptide, the TALE DNA-binding domain portion is able to bind itstarget site and/or its binding site, while the cleavage domain is ableto cleave DNA in the vicinity of the target site. With respect to afusion polypeptide in which a Cas DNA-binding domain is fused to anactivation domain, the Cas DNA-binding domain and the activation domainare in operative linkage if, in the fusion polypeptide, the CasDNA-binding domain portion is able to bind its target site and/or itsbinding site, while the activation domain is able to up-regulate geneexpression. When a fusion polypeptide in which a Cas DNA-binding domainis fused to a cleavage domain, the Cas DNA-binding domain and thecleavage domain are in operative linkage if, in the fusion polypeptide,the Cas DNA-binding domain portion is able to bind its target siteand/or its binding site, while the cleavage domain is able to cleave DNAin the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

DNA-Binding Domains

The methods described herein make use of compositions, for exampleHtt-modulating transcription factors, comprising a DNA-binding domainthat specifically binds to a target sequence in an Htt gene,particularly that bind to a mutant Htt allele comprising a plurality oftrinucleotide repeats. Any polynucleotide or polypeptide DNA-bindingdomain can be used in the compositions and methods disclosed herein, forexample DNA-binding proteins (e.g., ZFPs or TALEs) or DNA-bindingpolynucleotides (e.g., single guide RNAs). In certain embodiments, theDNA-binding domain binds to a target site comprising 9 to 28 (or anyvalue therebetween including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 or 27) contiguous nucleotides of SEQ ID NO:6.

In certain embodiments, the Htt-modulating transcription factor, or DNAbinding domain therein, comprises a zinc finger protein. Selection oftarget sites; ZFPs and methods for design and construction of fusionproteins (and polynucleotides encoding same) are known to those of skillin the art and described in detail in U.S. Pat. Nos. 6,140,081;5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453;6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO 98/53058; WO98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In certain embodiments, the ZFPs can bind selectively to either a mutantHtt allele or a wild-type Htt sequence. Htt target sites typicallyinclude at least one zinc finger but can include a plurality of zincfingers (e.g., 2, 3, 4, 5, 6 or more fingers). Usually, the ZFPs includeat least three fingers. Certain of the ZFPs include four, five or sixfingers, while some ZFPs include 8, 9, 10, 11 or 12 fingers. The ZFPsthat include three fingers typically recognize a target site thatincludes 9 or 10 nucleotides; ZFPs that include four fingers typicallyrecognize a target site that includes 12 to 14 nucleotides; while ZFPshaving six fingers can recognize target sites that include 18 to 21nucleotides. The ZFPs can also be fusion proteins that include one ormore regulatory domains, which domains can be transcriptional activationor repression domains. In some embodiments, the fusion protein comprisestwo ZFP DNA binding domains linked together. These zinc finger proteinscan thus comprise 8, 9, 10, 11, 12 or more fingers. In some embodiments,the two DNA binding domains are linked via an extendable flexible linkersuch that one DNA binding domain comprises 4, 5, or 6 zinc fingers andthe second DNA binding domain comprises an additional 4, 5, or 5 zincfingers. In some embodiments, the linker is a standard inter-fingerlinker such that the finger array comprises one DNA binding domaincomprising 8, 9, 10, 11 or 12 or more fingers. In other embodiments, thelinker is an atypical linker such as a flexible linker. The DNA bindingdomains are fused to at least one regulatory domain and can be thoughtof as a ‘ZFP-ZFP-TF’ architecture. Specific examples of theseembodiments can be referred to as “ZFP-ZFP-KOX” which comprises two DNAbinding domains linked with a flexible linker and fused to a KOXrepressor and “ZFP-KOX-ZFP-KOX” where two ZFP-KOX fusion proteins arefused together via a linker.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)JMol. Biol. 263:163-180; Argast et al. (1998)J Mol. Biol. 280:345-353 andthe New England Biolabs catalogue. In addition, the DNA-bindingspecificity of homing endonucleases and meganucleases can be engineeredto bind non-natural target sites. See, for example, Chevalier et al.(2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

“Two handed” zinc finger proteins are those proteins in which twoclusters of zinc finger DNA binding domains are separated by interveningamino acids so that the two zinc finger domains bind to twodiscontinuous target sites. An example of a two handed type of zincfinger binding protein is SIP1, where a cluster of four zinc fingers islocated at the amino terminus of the protein and a cluster of threefingers is located at the carboxyl terminus (see Remade et al, (1999)EMBO Journal 18 (18): 5073-5084). Each cluster of zinc fingers in theseproteins is able to bind to a unique target sequence and the spacingbetween the two target sequences can comprise many nucleotides.Two-handed ZFPs may include a functional domain, for example fused toone or both of the ZFPs. Thus, it will be apparent that the functionaldomain may be attached to the exterior of one or both ZFPs (see, FIG.1C) or may be positioned between the ZFPs (attached to both ZFPs) (see,FIG. 4).

Specific examples of Htt-targeted ZFPs are disclosed in U.S. PatentPublication No. 20130253040, which is incorporated by reference for allpurposes in its entirety herein, as well as in Table 1 below. The firstcolumn in this table is an internal reference name (number) for a ZFPand corresponds to the same name in column 1 of Table 2. “F” refers tothe finger and the number following “F” refers which zinc finger (e.g.,“F1” refers to finger 1).

TABLE 1  Htt-targeted zinc finger proteins Design SBS # F1 F2 F3 F4 F5F6 45643 QSGDLTR QSGDLTR QSGDLTR KHGNLSE KRCNLRC (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 1) NO: 1) NO: 1) NO: 2) NO: 3) 46025 CPSHLTR QSGDLTRKHGNLSE KRCNLRC RQFNRHQ (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 4)NO: 1) NO: 2) NO: 3) NO: 5)

The sequence and location for the target sites of these proteins aredisclosed in Table 2. Nucleotides in the target site that are contactedby the ZFP recognition helices are indicated in uppercase letters;non-contacted nucleotides indicated in lowercase.

TABLE 2  Target sites on human and mouse Htt SBS # Target Site 45643agCAGCAGcaGCAGCAGCAgcagcagca (SEQ ID NO: 6) 46025agCAGCAGCAGcaGCAGCAgcagcagca (SEQ ID NO: 6)

In certain embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector (TALE)DNA binding domain. See, e.g., U.S. Pat. No. 8,586,526, incorporated byreference in its entirety herein.

The plant pathogenic bacteria of the genus Xanthomonas are known tocause many diseases in important crop plants. Pathogenicity ofXanthomonas depends on a conserved type III secretion (T3 S) systemwhich injects more than 25 different effector proteins into the plantcell. Among these injected proteins are transcription activator-likeeffectors (TALE) which mimic plant transcriptional activators andmanipulate the plant transcriptome (see Kay et at (2007) Science318:648-651). These proteins contain a DNA binding domain and atranscriptional activation domain. One of the most well characterizedTALEs is AvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonaset at (1989) Mol Gen Genet 218: 127-136 and WO2010079430). TALEs containa centralized domain of tandem repeats, each repeat containingapproximately 34 amino acids, which are key to the DNA bindingspecificity of these proteins. In addition, they contain a nuclearlocalization sequence and an acidic transcriptional activation domain(for a review see Schornack S, et al (2006) J Plant Physiol 163(3):256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et at (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas.

Specificity of these TALEs depends on the sequences found in the tandemrepeats. The repeated sequence comprises approximately 102 bp and therepeats are typically 91-100% homologous with each other (Bonas et al,ibid). Polymorphism of the repeats is usually located at positions 12and 13 and there appears to be a one-to-one correspondence between theidentity of the hypervariable diresidues at positions 12 and 13 with theidentity of the contiguous nucleotides in the TALE's target sequence(see Moscou and Bogdanove (2009) Science 326:1501 and Boch et at (2009)Science 326:1509-1512). Experimentally, the code for DNA recognition ofthese TALEs has been determined such that an HD sequence at positions 12and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, C, Gor T, NN binds to A or G, and NG binds to T. These DNA binding repeatshave been assembled into proteins with new combinations and numbers ofrepeats, to make artificial transcription factors that are able tointeract with new sequences. In addition, U.S. Pat. No. 8,586,526 andU.S. Publication No. 20130196373, incorporated by reference in theirentireties herein, describe TALEs with N-cap polypeptides, C-cappolypeptides (e.g., +63, +231 or +278) and/or novel (atypical) RVDs.

Exemplary TALE are described in U.S. Patent Publication No. 20130253040,incorporated by reference in its entirety.

In certain embodiments, the DNA binding domains include a dimerizationand/or multimerization domain, for example a coiled-coil (CC) anddimerizing zinc finger (DZ). See, U.S. Patent Publication No.20130253040.

In still further embodiments, the DNA-binding domain comprises asingle-guide RNA of a CRISPR/Cas system, for example sgRNAs as disclosedin 20150056705.

Compelling evidence has recently emerged for the existence of anRNA-mediated genome defense pathway in archaea and many bacteria thathas been hypothesized to parallel the eukaryotic RNAi pathway (forreviews, see Godde and Bickerton, 2006. J. Mol. Evol. 62: 718-729;Lillestol et al., 2006. Archaea 2: 59-72; Makarova et al., 2006. Biol.Direct 1: 7.; Sorek et al., 2008. Nat. Rev. Microbiol. 6: 181-186).Known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi), the pathwayis proposed to arise from two evolutionarily and often physically linkedgene loci: the CRISPR (clustered regularly interspaced short palindromicrepeats) locus, which encodes RNA components of the system, and the cas(CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002.Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res.30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al.,2005. PLoS Comput. Biol. 1: e60). CRISPR loci in microbial hosts containa combination of CRISPR-associated (Cas) genes as well as non-coding RNAelements capable of programming the specificity of the CRISPR-mediatednucleic acid cleavage. The individual Cas proteins do not sharesignificant sequence similarity with protein components of theeukaryotic RNAi machinery, but have analogous predicted functions (e.g.,RNA binding, nuclease, helicase, etc.) (Makarova et al., 2006. Biol.Direct 1: 7). The CRISPR-associated (cas) genes are often associatedwith CRISPR repeat-spacer arrays. More than forty different Cas proteinfamilies have been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (E. coli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern,and Mtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

The Type II CRISPR, initially described in S. pyogenes, is one of themost well characterized systems and carries out targeted DNAdouble-strand break in four sequential steps. First, two non-coding RNA,the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA andmediates the processing of pre-crRNA into mature crRNAs containingindividual spacer sequences where processing occurs by a doublestrand-specific RNase III in the presence of the Cas9 protein. Third,the mature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. In addition,the tracrRNA must also be present as it base pairs with the crRNA at its3′ end, and this association triggers Cas9 activity. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation,’ (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system.

Type II CRISPR systems have been found in many different bacteria. BLASTsearches on publically available genomes by Fonfara et at ((2013) NucAcid Res 42(4):2377-2590) found Cas9 orthologs in 347 species ofbacteria. Additionally, this group demonstrated in vitro CRISPR/Cascleavage of a DNA target using Cas9 orthologs from S. pyogenes, S.mutans, S. therophilus, C. jejuni, N. meningitides, P. multocida and F.novicida. Thus, the term “Cas9” refers to an RNA guided DNA nucleasecomprising a DNA binding domain and two nuclease domains, where the geneencoding the Cas9 may be derived from any suitable bacteria.

The Cas9 protein has at least two nuclease domains: one nuclease domainis similar to a HNH endonuclease, while the other resembles a Ruvendonuclease domain. The HNH-type domain appears to be responsible forcleaving the DNA strand that is complementary to the crRNA while the Ruvdomain cleaves the non-complementary strand. The Cas 9 nuclease can beengineered such that only one of the nuclease domains is functional,creating a Cas nickase (see Jinek et al, ibid). Nickases can begenerated by specific mutation of amino acids in the catalytic domain ofthe enzyme, or by truncation of part or all of the domain such that itis no longer functional. Since Cas 9 comprises two nuclease domains,this approach may be taken on either domain. A double strand break canbe achieved in the target DNA by the use of two such Cas 9 nickases. Thenickases will each cleave one strand of the DNA and the use of two willcreate a double strand break.

The requirement of the crRNA-tracrRNA complex can be avoided by use ofan engineered “single-guide RNA” (sgRNA) that comprises the hairpinnormally formed by the annealing of the crRNA and the tracrRNA (seeJinek et at (2012) Science 337:816 and Cong et al (2013)Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineeredtracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the targetDNA when a double strand RNA:DNA heterodimer forms between the Casassociated RNAs and the target DNA. This system comprising the Cas9protein and an engineered sgRNA containing a PAM sequence has been usedfor RNA guided genome editing (see Ramalingam, ibid) and has been usefulfor zebrafish embryo genomic editing in vivo (see Hwang et at (2013)Nature Biotechnology 31 (3):227) with editing efficiencies similar toZFNs and TALENs.

The primary products of the CRISPR loci appear to be short RNAs thatcontain the invader targeting sequences, and are termed guide RNAs orprokaryotic silencing RNAs (psiRNAs) based on their hypothesized role inthe pathway (Makarova et al., 2006. Biol. Direct 1: 7; Hale et al.,2008. RNA, 14: 2572-2579). RNA analysis indicates that CRISPR locustranscripts are cleaved within the repeat sequences to release ^(˜)60-to 70-nt RNA intermediates that contain individual invader targetingsequences and flanking repeat fragments (Tang et al. 2002. Proc. Natl.Acad. Sci. 99: 7536-7541; Tang et al., 2005. Mol. Microbiol. 55:469-481; Lillestol et al. 2006. Archaea 2: 59-72; Brouns et al. 2008.Science 321: 960-964; Hale et al, 2008. RNA, 14: 2572-2579). In thearchaeon Pyrococcus furiosus, these intermediate RNAs are furtherprocessed to abundant, stable ^(˜)35-to 45-nt mature psiRNAs (Hale etal. 2008. RNA, 14: 2572-2579).

The requirement of the crRNA-tracrRNA complex can be avoided by use ofan engineered “single-guide RNA” (sgRNA) that comprises the hairpinnormally formed by the annealing of the crRNA and the tracrRNA (seeJinek et at (2012) Science 337:816 and Cong et al (2013)Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineeredtracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the targetDNA when a double strand RNA:DNA heterodimer forms between the Casassociated RNAs and the target DNA. This system comprising the Cas9protein and an engineered sgRNA containing a PAM sequence has been usedfor RNA guided genome editing (see Ramalingam ibid) and has been usefulfor zebrafish embryo genomic editing in vivo (see Hwang et at (2013)Nature Biotechnology 31 (3):227) with editing efficiencies similar toZFNs and TALENs.

Chimeric or sgRNAs can be engineered to comprise a sequencecomplementary to any desired target. In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. In some embodiments, the RNAs comprise 22 basesof complementarity to a target and of the form G[n19], followed by aprotospacer-adjacent motif (PAM) of the form NGG or NAG for use with aS. pyogenes CRISPR/Cas system. Thus, in one method, sgRNAs can bedesigned by utilization of a known ZFN target in a gene of interest by(i) aligning the recognition sequence of the ZFN heterodimer with thereference sequence of the relevant genome (human, mouse, or of aparticular plant species); (ii) identifying the spacer region betweenthe ZFN half-sites; (iii) identifying the location of the motif G[N20]GGthat is closest to the spacer region (when more than one such motifoverlaps the spacer, the motif that is centered relative to the spaceris chosen); (iv) using that motif as the core of the sgRNA. This methodadvantageously relies on proven nuclease targets. Alternatively, sgRNAscan be designed to target any region of interest simply by identifying asuitable target sequence the conforms to the G[n20]GG formula. Alongwith the complementarity region, an sgRNA may comprise additionalnucleotides to extend to tail region of the tracrRNA portion of thesgRNA (see Hsu et at (2013) Nature Biotech doi:10.1038/nbt.2647). Tailsmay be of +67 to +85 nucleotides, or any number therebetween with apreferred length of +85 nucleotides. Truncated sgRNAs may also be used,“tru-gRNAs” (see Fu et al, (2014) Nature Biotech 32(3): 279). Intru-gRNAs, the complementarity region is diminished to 17 or 18nucleotides in length.

Further, alternative PAM sequences may also be utilized, where a PAMsequence can be NAG as an alternative to NGG (Hsu 2014, ibid) using a S.pyogenes Cas9. Additional PAM sequences may also include those lackingthe initial G (Sander and Joung (2014) Nature Biotech 32(4):347). Inaddition to the S. pyogenes encoded Cas9 PAM sequences, other PAMsequences can be used that are specific for Cas9 proteins from otherbacterial sources. For example, the PAM sequences shown below (adaptedfrom Sander and Joung, ibid, and Esvelt et al, (2013) Nat Meth10(11):1116) are specific for these Cas9 proteins:

Species PAM S. pyogenes NGG S. pyogenes NAG S. mutans NGGS. thermophilius NGGNG S. thermophilius NNAAAW S. thermophilius NNAGAAS. thermophilius NNNGATT C. jejuni NNNNACA N. meningitides NNNNGATTP. multocida GNNNCNNA F. novicida NG

Thus, a suitable target sequence for use with a S. pyogenes CRISPR/Cassystem can be chosen according to the following guideline: [n17, n18,n19, or n20](G/A)G. Alternatively the PAM sequence can follow theguideline G[n17, n18, n19, n20](G/A)G. For Cas9 proteins derived fromnon-S. pyogenes bacteria, the same guidelines may be used where thealternate PAMs are substituted in for the S. pyogenes PAM sequences.

Most preferred is to choose a target sequence with the highestlikelihood of specificity that avoids potential off target sequences.These undesired off target sequences can be identified by consideringthe following attributes: i) similarity in the target sequence that isfollowed by a PAM sequence known to function with the Cas9 protein beingutilized; ii) a similar target sequence with fewer than three mismatchesfrom the desired target sequence; iii) a similar target sequence as inii), where the mismatches are all located in the PAM distal regionrather than the PAM proximal region (there is some evidence thatnucleotides 1-5 immediately adjacent or proximal to the PAM, sometimesreferred to as the ‘seed’ region (Wu et at (2014) Nature Biotechdoi:10.1038/nbt2889) are the most critical for recognition, so putativeoff target sites with mismatches located in the seed region may be theleast likely be recognized by the sg RNA); and iv) a similar targetsequence where the mismatches are not consecutively spaced or are spacedgreater than four nucleotides apart (Hsu 2014, ibid). Thus, byperforming an analysis of the number of potential off target sites in agenome for whichever CRIPSR/Cas system is being employed, using thesecriteria above, a suitable target sequence for the sgRNA may beidentified.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. In some aspects, a functionalderivative may comprise a single biological property of a naturallyoccurring Cas protein. In other aspects, a function derivative maycomprise a subset of biological properties of a naturally occurring Casprotein. Suitable derivatives of a Cas polypeptide or a fragment thereofinclude but are not limited to mutants, fusions, covalent modificationsof Cas protein or a fragment thereof. Cas protein, which includes Casprotein or a fragment thereof, as well as derivatives of Cas protein ora fragment thereof, may be obtainable from a cell or synthesizedchemically or by a combination of these two procedures. The cell may bea cell that naturally produces Cas protein, or a cell that naturallyproduces Cas protein and is genetically engineered to produce theendogenous Cas protein at a higher expression level or to produce a Casprotein from an exogenously introduced nucleic acid, which nucleic acidencodes a Cas that is same or different from the endogenous Cas. In somecase, the cell does not naturally produce Cas protein and is geneticallyengineered to produce a Cas protein.

Exemplary CRISPR/Cas nuclease systems targeted to specific genes aredisclosed for example, in U.S. Publication No. 20150056705.

Thus, the nuclease comprises a DNA-binding domain in that specificallybinds to a target site in any gene into which it is desired to insert adonor (transgene) in combination with a nuclease domain that cleavesDNA.

Fusion Molecules

The DNA-binding domains may be fused to any additional molecules (e.g.,polypeptides) for use in the methods described herein. In certainembodiments, the methods employ fusion molecules comprising at least oneDNA-binding molecule (e.g., ZFP, TALE or single guide RNA) and aheterologous regulatory (functional) domain (or functional fragmentthereof).

In certain embodiments, the functional domain comprises atranscriptional regulatory domain. Common domains include, e.g.,transcription factor domains (activators, repressors, co-activators,co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max,mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymesand their associated factors and modifiers; DNA rearrangement enzymesand their associated factors and modifiers; chromatin associatedproteins and their modifiers (e.g. kinases, acetylases anddeacetylases); and DNA modifying enzymes (e.g., methyltransferases,topoisomerases, helicases, ligases, kinases, phosphatases, polymerases,endonucleases) and their associated factors and modifiers. See, e.g.,U.S. Publication No. 20130253040, incorporated by reference in itsentirety herein.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipelet al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol.Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol.23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci.25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRABl. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al.(1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; andRobertson et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al.(2000) Plant J. 22:19-27.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin). Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

The fusion molecule may be formulated with a pharmaceutically acceptablecarrier, as is known to those of skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned WO00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

In certain embodiments, the fusion protein comprises a DNA-bindingdomain and a nuclease domain to create functional entities that are ableto recognize their intended nucleic acid target through their engineered(ZFP or TALE) DNA binding domains and create nucleases (e.g., zincfinger nuclease or TALE nucleases) cause the DNA to be cut near the DNAbinding site via the nuclease activity.

Thus, the methods and compositions described herein are broadlyapplicable and may involve any nuclease of interest. Non-limitingexamples of nucleases include meganucleases, TALENs and zinc fingernucleases. The nuclease may comprise heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; TALENs; meganucleaseDNA-binding domains with heterologous cleavage domains) or,alternatively, the DNA-binding domain of a naturally-occurring nucleasemay be altered to bind to a selected target site (e.g., a meganucleasethat has been engineered to bind to site different than the cognatebinding site).

The nuclease domain may be derived from any nuclease, for example anyendonuclease or exonuclease. Non-limiting examples of suitable nuclease(cleavage) domains that may be fused to Htt DNA-binding domains asdescribed herein include domains from any restriction enzyme, forexample a Type IIS Restriction Enzyme (e.g., FokI). In certainembodiments, the cleavage domains are cleavage half-domains that requiredimerization for cleavage activity. See, e.g., U.S. Pat. Nos. 8,586,526;8,409,861 and 7,888,121, incorporated by reference in their entiretiesherein. In general, two fusion proteins are required for cleavage if thefusion proteins comprise cleavage half-domains. Alternatively, a singleprotein comprising two cleavage half-domains can be used. The twocleavage half-domains can be derived from the same endonuclease (orfunctional fragments thereof), or each cleavage half-domain can bederived from a different endonuclease (or functional fragments thereof).In addition, the target sites for the two fusion proteins are preferablydisposed, with respect to each other, such that binding of the twofusion proteins to their respective target sites places the cleavagehalf-domains in a spatial orientation to each other that allows thecleavage half-domains to form a functional cleavage domain, e.g., bydimerizing.

The nuclease domain may also be derived any meganuclease (homingendonuclease) domain with cleavage activity may also be used with thenucleases described herein, including but not limited to I-SceI, I-CeuI,PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-TevI, I-TevII and I-TevIII.

In certain embodiments, the nuclease comprises a compact TALEN (cTALEN).These are single chain fusion proteins linking a TALE DNA binding domainto a TevI nuclease domain. The fusion protein can act as either anickase localized by the TALE region, or can create a double strandbreak, depending upon where the TALE DNA binding domain is located withrespect to the meganuclease (e.g., TevI) nuclease domain (see Beurdeleyet at (2013) Nat Comm: 1-8 DOI: 10.1038/ncomms2782).

In other embodiments, the TALE-nuclease is a mega TAL. These mega TALnucleases are fusion proteins comprising a TALE DNA binding domain and ameganuclease cleavage domain. The meganuclease cleavage domain is activeas a monomer and does not require dimerization for activity. (SeeBoissel et al., (2013) Nucl Acid Res: 1-13, doi: 10.1093/nar/gkt1224).

In addition, the nuclease domain of the meganuclease may also exhibitDNA-binding functionality. Any TALENs may be used in combination withadditional TALENs (e.g., one or more TALENs (cTALENs or FokI-TALENs)with one or more mega-TALs) and/or ZFNs.

In addition, cleavage domains may include one or more alterations ascompared to wild-type, for example for the formation of obligateheterodimers that reduce or eliminate off-target cleavage effects. See,e.g., U.S. Pat. Nos. 7,914,796; 8,034,598; and 8,623,618, incorporatedby reference in their entireties herein.

Nucleases as described herein may generate double- or single-strandedbreaks in a double-stranded target (e.g., gene). The generation ofsingle-stranded breaks (“nicks”) is described, for example in U.S. Pat.No. 8,703,489, incorporated herein by reference which describes howmutation of the catalytic domain of one of the nucleases domains resultsin a nickase.

Thus, a nuclease (cleavage) domain or cleavage half-domain can be anyportion of a protein that retains cleavage activity, or that retains theability to multimerize (e.g., dimerize) to form a functional cleavagedomain.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in U.S. Publication No.20090111119. Nuclease expression constructs can be readily designedusing methods known in the art.

Expression of the fusion proteins may be under the control of aconstitutive promoter or an inducible promoter, for example thegalactokinase promoter which is activated (de-repressed) in the presenceof raffinose and/or galactose and repressed in presence of glucose. Incertain embodiments, the promoter self-regulates expression of thefusion protein, for example via inclusion of high affinity bindingsites. See, e.g., U.S. Application No. 61/955,002, filed Mar. 18, 2014.

Delivery

The proteins and/or polynucleotides (e.g., Htt repressors) andcompositions comprising the proteins and/or polynucleotides describedherein may be delivered to a target cell by any suitable meansincluding, for example, by injection of proteins, via mRNA and/or usingan expression construct (e.g., plasmid, lentiviral vector, AAV vector,Ad vector, etc.). In preferred embodiments, the repressor is deliveredusing AAV9.

Methods of delivering proteins comprising zinc finger proteins asdescribed herein are described, for example, in U.S. Pat. Nos.6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, thedisclosures of all of which are incorporated by reference herein intheir entireties.

Any vector systems may be used including, but not limited to, plasmidvectors, retroviral vectors, lentiviral vectors, adenovirus vectors,poxvirus vectors; herpesvirus vectors and adeno-associated virusvectors, etc. See, also, U.S. Pat. Nos. 8,586,526; 6,534,261; 6,607,882;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporatedby reference herein in their entireties. Furthermore, it will beapparent that any of these vectors may comprise one or more DNA-bindingprotein-encoding sequences. Thus, when one or more Htt repressors areintroduced into the cell, the sequences encoding the protein componentsand/or polynucleotide components may be carried on the same vector or ondifferent vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple Htt repressors orcomponents thereof

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered Htt repressors in cells(e.g., mammalian cells) and target tissues. Such methods can also beused to administer nucleic acids encoding such repressors (or componentsthereof) to cells in vitro. In certain embodiments, nucleic acidsencoding the repressors are administered for in vivo or ex vivo genetherapy uses. Non-viral vector delivery systems include DNA plasmids,naked nucleic acid, and nucleic acid complexed with a delivery vehiclesuch as a liposome or poloxamer. Viral vector delivery systems includeDNA and RNA viruses, which have either episomal or integrated genomesafter delivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds.) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,naked RNA, artificial virions, and agent-enhanced uptake of DNA.Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can alsobe used for delivery of nucleic acids. In a preferred embodiment, one ormore nucleic acids are delivered as mRNA. Also preferred is the use ofcapped mRNAs to increase translational efficiency and/or mRNA stability.Especially preferred are ARCA (anti-reverse cap analog) caps or variantsthereof. See US patents U.S. Pat. No. 7,074,596 and U.S. Pat. No.8,153,773, incorporated by reference herein.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet at (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs, TALEs or CRISPR/Cas systems takeadvantage of highly evolved processes for targeting a virus to specificcells in the body and trafficking the viral payload to the nucleus.Viral vectors can be administered directly to patients (in vivo) or theycan be used to treat cells in vitro and the modified cells areadministered to patients (ex vivo). Conventional viral based systems forthe delivery of ZFPs, TALEs or CRISPR/Cas systems include, but are notlimited to, retroviral, lentivirus, adenoviral, adeno-associated,vaccinia and herpes simplex virus vectors for gene transfer. Integrationin the host genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmouse leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6, AAV8AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such asAAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with thepresent invention. In preferred embodiments, AAV9 is used.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney mouse leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion, including direct injection into the brain) or topicalapplication, as described below. Alternatively, vectors can be deliveredto cells ex vivo, such as cells explanted from an individual patient(e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universaldonor hematopoietic stem cells, followed by reimplantation of the cellsinto a patient, usually after selection for cells which haveincorporated the vector.

In certain embodiments, the compositions as described herein (e.g.,polynucleotides and/or proteins) are delivered directly in vivo. Thecompositions (cells, polynucleotides and/or proteins) may beadministered directly into the central nervous system (CNS), includingbut not limited to direct injection into the brain or spinal cord. Oneor more areas of the brain may be targeted, including but not limitedto, the hippocampus, the substantia nigra, the nucleus basalis ofMeynert (NBM), the striatum and/or the cortex. Alternatively or inaddition to CNS delivery, the compositions may be administeredsystemically (e.g., intravenous, intraperitoneal, intracardial,intramuscular, intrathecal, subdermal, and/or intracranial infusion).Methods and compositions for delivery of compositions as describedherein directly to a subject (including directly into the CNS) includebut are not limited to direct injection (e.g., stereotactic injection)via needle assemblies. Such methods are described, for example, in U.S.Pat. Nos. 7,837,668; 8,092,429, relating to delivery of compositions(including expression vectors) to the brain and U.S. Patent PublicationNo. 20060239966, incorporated herein by reference in their entireties.

The effective amount to be administered will vary from patient topatient and according to the mode of administration and site ofadministration. Accordingly, effective amounts are best determined bythe physician administering the compositions and appropriate dosages canbe determined readily by one of ordinary skill in the art. Afterallowing sufficient time for integration and expression (typically 4-15days, for example), analysis of the serum or other tissue levels of thetherapeutic polypeptide and comparison to the initial level prior toadministration will determine whether the amount being administered istoo low, within the right range or too high. Suitable regimes forinitial and subsequent administrations are also variable, but aretypified by an initial administration followed by subsequentadministrations if necessary. Subsequent administrations may beadministered at variable intervals, ranging from daily to annually toevery several years. In certain embodiments, when using a viral vectorsuch as AAV, the dose administered is between 1×10¹⁰ and 5×10¹⁵ vg/ml(or any value therebetween), even more preferably between 1×10¹¹ and1×10¹⁴ vg/ml (or any value therebetween), even more preferably between1×10¹² and 1×10¹³ vg/ml (or any value therebetween).

To deliver ZFPs using adeno-associated viral (AAV) vectors directly tothe human brain, a dose range of 1×10¹⁰-5×10¹⁵ (or any valuetherebetween, including for example between 1×10¹¹ and 1×10¹⁴ vg/ml or1×10¹² and 1×10¹³ vg/ml) vector genome per striatum can be applied. Asnoted, dosages may be varied for other brain structures and fordifferent delivery protocols. Methods of delivering AAV vectors directlyto the brain are known in the art. See, e.g., U.S. Pat. Nos. 9,089,667;9,050,299; 8,337,458; 8,309,355; 7,182,944; 6,953,575; and 6,309,634.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with at leastone Htt repressor or component thereof and re-infused back into thesubject organism (e.g., patient). In a preferred embodiment, one or morenucleic acids of the Htt repressor are delivered using AAV9. In otherembodiments, one or more nucleic acids of the Htt repressor aredelivered as mRNA. Also preferred is the use of capped mRNAs to increasetranslational efficiency and/or mRNA stability. Especially preferred areARCA (anti-reverse cap analog) caps or variants thereof. See U.S. Pat.Nos. 7,074,596 and 8,153,773, incorporated by reference herein in theirentireties. Various cell types suitable for ex vivo transfection arewell known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+(T cells), CD45+ (panB cells), GR-1 (granulocytes),and Iad (differentiated antigen presenting cells) (see Inaba et al., J.Exp. Med. 176:1693-1702 (1992)).

Stem cells that have been modified may also be used in some embodiments.For example, neuronal stem cells that have been made resistant toapoptosis may be used as therapeutic compositions where the stem cellsalso contain the ZFP TFs of the invention. Resistance to apoptosis maycome about, for example, by knocking out BAX and/or BAK using BAX- orBAK-specific TALENs or ZFNs (see, U.S. Pat. No. 8,597,912) in the stemcells, or those that are disrupted in a caspase, again using caspase-6specific ZFNs for example. These cells can be transfected with the ZFPTFs or TALE TFs that are known to regulate mutant or wild-type Htt.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Naldini et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull etal. (1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0,5P2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pischia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used. In a preferredembodiment, the methods and composition are delivered directly to abrain cell, for example in the striatum.

Applications

Htt-binding molecules (e.g., ZFPs, TALEs, CRISPR/Cas systems, Ttago,etc.) as described herein, and the nucleic acids encoding them, can beused for a variety of applications. These applications includetherapeutic methods in which a Htt-binding molecule (including a nucleicacid encoding a DNA-binding protein) is administered to a subject (e.g.,an AAV such as AAV9) and used to modulate the expression of a targetgene within the subject. The modulation can be in the form ofrepression, for example, repression of mHtt that is contributing to anHD disease state. Alternatively, the modulation can be in the form ofactivation when activation of expression or increased expression of anendogenous cellular gene can ameliorate a diseased state. In stillfurther embodiments, the modulation can be cleavage (e.g., by one ormore nucleases), for example, for inactivation of a mutant Htt gene. Asnoted above, for such applications, the Htt-binding molecules, or moretypically, nucleic acids encoding them are formulated with apharmaceutically acceptable carrier as a pharmaceutical composition.

The Htt-binding molecules, or vectors encoding them, alone or incombination with other suitable components (e.g. liposomes,nanoparticles or other components known in the art), can be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. Formulations suitable for parenteraladministration, such as, for example, by intravenous, intramuscular,intradermal, and subcutaneous routes, include aqueous and non-aqueous,isotonic sterile injection solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. Compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, intracranially or intrathecally. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials. Injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The dose administered to a patient should be sufficient to effect abeneficial therapeutic response in the patient over time. The dose isdetermined by the efficacy and K_(d) of the particular Htt-bindingmolecule employed, the target cell, and the condition of the patient, aswell as the body weight or surface area of the patient to be treated.The size of the dose also is determined by the existence, nature, andextent of any adverse side-effects that accompany the administration ofa particular compound or vector in a particular patient.

Beneficial therapeutic response can be measured in a number of ways. Forexample, improvement in Huntington's associates movement disorders suchas involuntary jerking or writhing movements, muscle problems, such asrigidity or muscle contracture (dystonia), slow or abnormal eyemovements, impaired gait, posture and balance, difficulty with thephysical production of speech or swallowing and the impairment ofvoluntary movements can be measured. Other impairments, such ascognitive and psychiatric disorders can also be monitored for signs ofimprovement associated with treatment. The UHDRS scale can be used toquantitate clinical features of the disease.

For patients that are pre-symptomatic, treatment can be especiallyimportant because it affords the opportunity to treat the disease priorto the extensive neurodegeneration that occurs in HD. This damageinitiates prior to the development of the overt symptoms describedabove. HD pathology primarily involves the toxic effect of mutant Htt instriatal medium spiny neurons. These medium spiny neurons express highlevels of phosphodiesterase 10A (PDE10A) which regulates cAMP and cGMPsignaling cascades that are involved in gene transcription factors,neurotransmitter receptors and voltage-gated channels (Niccolini et at(2015) Brain 138:3016-3029), and it has been shown that the expressionof PDE10A is reduced in HD mice and post-mortem studies in humans foundthe same. Recently, positron emission tomography (PET) ligands have beendeveloped that are ligands for the PDE10A enzyme (e.g. ¹¹C-IMA107,(Niccolini et al, ibid; ¹⁸FMNI-659 (Russell et at (2014) JAMA Neurol71(12):1520-1528), and these molecules have been used to evaluatepre-symptomatic HD patients. The studies have been shown that PDE10Alevels are altered in HD patients even before symptoms develop. Thus,evaluation of PDE10A levels by PET can be done before, during and aftertreatment to measure therapeutic efficacy of the compositions of theinvention. “Therapeutic efficacy” can mean improvement of clinical andmolecular measurements, and can also mean protecting the patient fromany further decreases in medium spiny neuron function or an increase inspiny neuron loss, or from further development of the overt clinicalpresentations associated with HD.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the Htt-modulator comprises a zinc finger protein.It will be appreciated that this is for purposes of exemplification onlyand that other Htt-modulators (e.g., repressors) can be used, including,but not limited to, TALE-TFs, a CRISPR/Cas system, additional ZFPs,ZFNs, TALENs, additional CRISPR/Cas systems (e.g., Cfp systems), homingendonucleases (meganucleases) with engineered DNA-binding domains.

EXAMPLES Example 1: Htt Repressors

Zinc finger proteins 45643 and 46025 (see Table 1) targeted to Htt wereengineered essentially as described in U.S. Pat. No. 6,534,261; U.S.Patent Publication Nos. 20150056705; 20110082093; 20130253040; and U.S.application Ser. No. 14/706,747. Table 1 shows the recognition helicesof the DNA binding domain of these ZFPs, while Table 2 shows the targetsequences of these ZFPs. The ZFPs were evaluated and shown to be bind totheir target sites.

ZFPs 45643 and 46025 were operably linked to a KRAB repression domain toform ZFP-TF that repress Htt. The ZFP TFs were transfected into humancells (e.g., cells derived from HD patients) and expression of Htt wasmonitored using real-time RT-PCR. Both ZFP-TFs were found to beeffective in selectively repressing mutant Htt expression. ZFP-TFs arefunctional repressors when formulated as plasmids, in mRNA form, in Advectors, lentiviral vectors and/or in AAV vectors (e.g., AAV9).

Example 2: Materials and Methods Animals

Two rhesus monkeys (Macaca mulatta, 4-15 years of age, >4 kg) wereincluded in this study. Experiments were performed according to NationalInstitutes of Health guidelines and to protocols approved by theInstitutional Animal Care and Use Committee at University of CaliforniaSan Francisco.

Vector Preparation.

AAV9-containing green fluorescent protein (GFP) under the control of thecytomegalovirus promoter was generated by triple transfection of HEK-293cells as previously described in Matsushita et al. (1998) Gene Ther 5:938-945. AAV9-GFP was diluted immediately before use to a concentrationof 1.4×10¹³ vg/ml (high dose) or 1.4×10¹² vg/ml (low dose) inphosphate-buffered saline and 0.001% (vol/vol) Pluronic F-68.

Surgery and Vector Infusion.

Each NHP underwent stereotactic placement of skull-mounted,MR-compatible temporary plastic plugs. The animal was then placed supinein an MRI-compatible stereotactic frame. After craniectomy, thecannula-guides were secured to the skull over both hemispheres. Afterplacement of the plugs, the intubated animal was moved to the platformtable in the MM suite and placed on inhaled isoflurane (1-3%). Guidesunder sterile conditions were filled with MR-visible tracer (Prohance,Singem, Germany) to localize the plugs in the MR images to calculate thetrajectory to the target structures inside the brain. Then, the NHP wasmoved into the MR magnet and high-resolution anatomical MR scan wasacquired for target identification and surgical planning. After thetarget was selected, a custom-designed, ceramic, fused silicareflux-resistant cannula with a 3-mm stepped tip was used for vectorinfusion as described previously in Richardson et al. (2011) Mol Ther19: 1048-1057; Krauze et al. (2005)J Neurosurg 103: 923-929; andFiandaca et al. (2008). Neurotherapeutics 5: 123-127.

The cannula was attached to a 1-ml syringe mounted onto anMRI-compatible infusion pump (Harvard Apparatus, Boston, Mass.). Theinfusion initiated at 1 μl/min, and after visualizing the infusate atthe cannula tip, the cannula was introduced through the guide-stem intothe brain. When the depth-stop encountered the top of the guide-stem, itwas secured with a locking screw. The infusion rate was ramped up froman initial 1 μl/min to a final 5 μl/min. Each NHP received infusionscovering the pre- and post-commissural putamen simultaneously in eachputamen (bilateral). The total infusion volume per hemisphere was 100 μlin both hemispheres. Once the infusion ended, guide-devices were removedfrom the skull, and animals were returned to their home cages andmonitored during recovery from anesthesia.

MRI Acquisition.

Animals were scanned on a Siemens Verio Magnetom 3.0T MRI (Siemens,Malvern, Pa.). T1-weighted fast low angle shot (FLASH) acquisitionsobtained with a 4° flip angle on the first scan produced aproton-density weighted image to trace gadolinium at the cannula tip (8ms TE, 28 ms TR, 3 excitations, 256×3×192 matrix, 14×14 mm field ofview, 1 mm slices). All subsequent scans were serially acquired at a 40°flip angle to increase the T1-weighting and highlight the Gd signalenhancement.

Tissue Processing.

Animals infused with AAV9 were perfused transcardially approximately 3weeks after AAV-GFP infusion with cold saline followed by 4%paraformaldehyde. Brains were harvested and histologically analyzedusing previously established methods. In short, 6 mm coronal blocks werecollected via a brain matrix and immediately post-fixed inparaformaldehyde overnight, then cryoprotected in 30% (w/v) sucrose thefollowing day. A sliding microtome was used to cut 40-μm serialsections. Chromagenic staining was performed on free-floating sectionsto visualize GFP expression by methods we have previously established.

Immunohistochemistry.

For each serotype, sections were collected in sequence, stored in100-well containers in cryoprotectant solution (0.5 M sodium phosphatebuffer, pH 7.4, 30% glycerol, and 30% ethylene glycol) at 4° C. untilfurther processing. Immunohistochemistry was performed on free-floatingsections. Briefly, washes with PBS for the horseradish peroxidase(HRP)-based procedure, or in PBS with 0.1% Tween 20 (PBST) forfluorescence staining, were performed between each immunohistochemicalstep. Endogenous peroxidase activity (for peroxidase-based procedures)was quenched for 30 minutes at room temperature. Blocking ofnon-specific staining was accomplished by incubation of sections in 20%horse serum in PBST for 60 min at room temperature. Thereafter, sectionswere incubated overnight with specific primary antibodies. Primaryantibodies used in the immunohistochemical procedures were as follows:polyclonal rabbit anti-Iba1, PAb, 1:500 (www.biocare.net/); monoclonalmouse and anti-GFAP, 1:10,000 for HRP-based staining and polyclonalrabbit and anti-GFAP 1:1000 for fluorescence (www.millipore.com);monoclonal mouse anti-NeuN, 1:5,000 for HRP-based staining and 1:500 forfluorescence staining (Millipore); monoclonal and mouse anti-TH, 1:1000(Millipore, MAB318); polyclonal mouse and monoclonal rabbit anti-GFP,1:200 and 1:500, respectively, (Life Technologies; Millipore). Allantibodies were dissolved in Da Vinci diluent (Biocare). After threerinses in PBS for 5 min at room temperature, sections for HRP-basedstaining were incubated with either Mach 2 anti-mouse HRP polymer(Biocare) or March 2 anti-rabbit HRP polymer (Biocare) for 1 h at roomtemperature. The activity of bound HRP was visualized by means of acommercially available kit with 3,3′-diaminobenzidine peroxide substrate(Vectro Labs). NeuN-stained sections were counterstained with CresylViolet stain. Finally, immunostained sections were mounted ongelatinized slides, dehydrated in alcohol and xylene and cover-slippedwith Cytoseal™ (Fisher Scientific).

For dual fluorescent immunostaining of different antigens (GFP/GFAP,GFP/NeuN, GFP/TH, and GFP/Iba1), a combination of primary antibodies wasapplied to sections as a cocktail of primary antibodies by overnightincubation at 4° C. All primary antibodies were dissolved in DaVincidiliuent (Biocare). After three washes in PBST, monoclonal primaryantibodies were visualized by incubation in the dark for 2 hours withappropriate secondary fluorochrome-conjugated antibodies: goatanti-mouse DyLight 549 (red) (Biocare), goat anti-rabbit DyLight 549,donkey anti-rabbit Alexa Fluor 555 (Life Technologies), goat anti-mouseDyLight 488 (green), goat anti-rabbit DyLight 488, and donkeyanti-rabbit Alexa Fluor 488. All secondary antibodies were dissolved at1:1,000 dilution in Fluorescence Antibody Diluent (Biocare). Sectionswere cover-slipped with Vectashield Hard Set, Mounting Medium forFluorescence (Vector Labs). Control sections were processed withoutprimary antibodies, and no significant immunostaining was observed underthese conditions.

Semi-Quantitative Analyses.

Distribution volume (Vd) analysis was performed with Brainlab iPlan FlowSuite (Brainlab, Munich, Germany; www.brainlab.com). Infusion sites,cannula tracts and cannula tip were identified on T1-weighted MR imagesin the coronal, axial, and sagittal planes. Regions of interest (ROIs)were delineated to outline T1 gadolinium signal and target putamen.Three-dimensional volumetric reconstructions of the image series and ROIwere analyzed to determine estimated Vd of infusions and its ratio withrespect to the total volume of infusate (Vi).

Analysis of Brain Sections.

All processed sections were examined and digitally photographed on aZeiss Axioskop microscope (Zeiss) equipped with CCD color video cameraand image analysis system (Axiovision Software; Zeiss). For each monkey,the number of GFP-positive and NeuN-positive cells was determined inboth hemispheres from coronal sections through the putamen. Fluorescencemicroscopy was used to determine the number of double-labeled cells insections. Photomicrographs for double-labeled sections were obtained bymerging images from two separate channels (red-rhodamine andgreen-fluorescein isothiocyanate; co-localization appears yellow)without altering the position of the sections or focus (objective×20 and×40, Carl Zeiss microscopy with ApoTome mode). For each double staining,sections were selected anterior and posterior at ˜500-μm distance fromthe site of injection. To identify the proportion of cells expressingGFP/NeuN, GFP/GFAP, GFP/TH and GFP/Iba1, each section was analyzed byfirst using one channel for the presence of phenotype-specific cells(TH, GFAP, Iba1, or NeuN) and second a combined channel for the numberof co-stained cells.

Sections stained for NeuN and GFP were used for counting from threesections at three different levels of the injection site in the putamen(bilateral). NeuN positive and GFP positive cells were counted at200-fold magnification from 5 randomly acquired frames (350 μm²) in boththe transduced area. In the non-transduced area, 5 randomized frameswere taken at a distance of 350 μm from the defined border ofexpression. These analyses permitted quantitative comparisons of thevectors, although they do not reflect the total number of transducedcells in vivo. Cell counts in each sampled region were averaged acrosssections for each animal and the final data are presented as the meannumber of NeuN positive and GFP positive.

Example 3: Infusion and Transduction Efficiency

We have shown previously that AAV2 is a neurotropic vector that istransported in an anterograde direction along neurons when it is infusedinto the parenchyma of rat and non-human primate (NHP) brain. See, e.g.,Ciesielska et al. (2011). Mol Ther 19: 922-927; Kells et al. (2012)Neurobiol Dis 48: 228-235. This transport of intact viral particles issufficiently robust that vector is apparently released from projectingnerve terminals where it is able to transduce distal neurons. Thus,infusion of AAV2 into NHP thalamus resulted in robust transduction ofcortical neurons contained entirely within the cortex. In contrast, AAV6is transported along axons in a retrograde direction and is almost asneurotropic as is AAV2. See, e.g., Salegio et al. (2012) Gene Ther.20(3):348-52; San Sebastian et al. (2013) Gene Ther 20: 1178-1183. Forexample, transduction of NHP putamen results in transgene expression ofcortico-striatal neurons.

In this study, 2 NHPs received putaminal infusions of AAV9-GFP at eithera high dose (HD; left hemisphere; 1.5×10¹³ vg/mL) or a low dose (LD;right hemisphere; 1.5×10¹² vg/mL). The post-surgical in-life phase wasintentionally kept short (3 weeks) in order to limit potential confoundsarising from cell-mediated responses to GFP as previously described inCiesielska et al. (2013) Mol Ther 21: 158-166; Samaranch et al. (2014)Mol Ther 22: 329-337.

The distribution of gadolinium-enhanced signal on MRI was evaluatedvolumetrically as previously described in Richardson et al. (2011)Stereotact Funct Neurosurg 89:141-151. Results are shown in FIG. 1. Itshould be noted that the approximate shape of the primate putamen issomewhat conical in sagittal orientation with the broader cross-sectionat the anterior end narrowing towards the posterior portion. Coverage ofthe putamen by MRI contrast reagent was, however, almost complete. Theoverlap between GFP and gadolinium signal indicated that the infusatewas well-contained with little leakage in the anterior and medialportions of the putamen, and expanded over an area three-fold that ofthe MR image contrast area. Immunohistochemical staining for GFPexpression was outlined and superimposed against an outline of putamenreconstructed from the baseline sequence of MR images at various coronallevels within the spatial bounds of the infusion. This is quitedifferent from what has been seen with AAV2 where expression oftransgene correlated almost exactly with the MRI signal (Fiandaca et al.(2009) Neuroimage 47 Suppl 2: T27-35).

Furthermore, robust reporter expression was evident both in HD and in LDputamina with an abundance of cell bodies and neuronal fibers (FIGS. 2Athrough 2D). We counted GFP+/NeuN+ neurons by immunofluorescencethroughout the primary area of transduction (PAT; mm²) in the plane ofthe cannula tract, based on previously described methods (Ciesielska etal. (2013) Mol Ther 21:158-166). The intensity of GFP-positive neuronalexpression diminished promptly along a distinct perimeter ≦350 μm fromtransduced areas, hereafter described as “outside” the PAT (oPAT). Inthis part of the study, we performed counted NeuN+ cell bodies in thePAT and oPAT, as well as in putaminal sections processed from twohealthy, naïve monkeys in order to ascertain vector-dependent effects.

We further analyzed whether efficiency of transduction at the PAT andoPAT was dependent upon dose in terms of NeuN-positive and GFP-positivelabels that appear to reveal AAV9-mediated transduction and GFPexpression in neuronal cell bodies. We saw no evidence of any neuronalloss due to AAV9-GFP transduction, consistent with our previousobservations of a relatively slow build-up of anti-GFP responses evidentmore than 6 weeks after AAV9-GFP infusion but not at 3 weeks, althoughwe saw evidence of activation of microglia and MHC-II upregulation(FIGS. 2E through 2H).

There are a number of mechanisms for antigen-presentation in the brain,chiefly astrocytes (Cornet et al. (2000) J Neuroimmunol 106:69-77 andmicroglia (Nelson et al. (2002) Ann Med 34:491-500). To determine thecellular specificity of AAV9 for neuronal and glial targets at theinfusion site, we immunostained brain sections for the transgene andcell-specific markers, including NeuN neuronal marker for neurons, glialfibrillary acid protein (GFAP) for astrocytes, and Iba1, amicroglia-specific marker.

As shown in FIG. 3, double immunofluorescence staining against GFP witheach of the cellular markers revealed transgene was readily expressed inboth neurons and astrocytes, whereas microglia were not transduceddespite massive GFP expression in neighboring cell bodies and neuronalfibers. Nevertheless, activation of microglia in transduced regions wasreadily observable (FIGS. 2E through 2H), indicative of the ability ofmicroglia to sense the innate immune status of their local environment.

Example 4: Axonal Transport

Infusion of AAV9-GFP into putamen yielded transduction in distalstructures. GFP staining was observed, for example, in cell bodies ofthe prefrontal, frontal, and parietal cortex (FIG. 4). GFP-positive cellbodies and fibers were also present in thalamus and components of thebasal ganglia, including the substantia nigra pars compacta (SNc) andpars reticulata (SNr), as well as subthalamic nucleus (STN) and infibers of the medial forebrain bundle (MFB) (FIG. 5). There was a strongvector dose-dependence of axonal transport to distal loci (FIG. 7).Thus, the high but not low dose of AAV9 resulted in cell body labelingin STN despite the fact that there is no direct neuronal connectionbetween the putamen and STN. We have previously noted such indirectanterograde transport within basal ganglia with AAV2 (Ciesielska et at(2011) Mol Ther 19:922-927, and Kells (2012), ibid and from thalamus tocortex (Kells et al. (2009) Proc Natl Acad Sci USA 106: 2407-2411).However, the significant dose-dependence of AAV9-GFP transport implies a2-stage transport of vector first to the globus pallidus and then toSTN. Anterograde transport of AAV9 was further supported by the presenceof GFP-positive cell bodies within the substantia nigra pars reticulata(SNr) with the presence of fibers marking neurons projecting into SNrfrom putamen (FIG. 6). Surprisingly, cell bodies in substantia nigrapars compacta (SNc) were also GFP-positive, indicative of retrogradetransport from putamen to which SNc sends highly arborized projections.Although not as dramatic a difference as with STN, there was a cleardose effect of AAV9-GFP on transduction in these areas.

Thus, we conclude that AAV9 is transported along axons in bothdirections, a phenomenon that accounts at least partly for theremarkable distribution of AAV9 in the primate brain. AAV9 differedstrikingly from AAV2 and AAV6 in terms of axonal transport and cell-typespecificity. AAV9 transduced astrocytes and neurons but not microglia.The vector was transported axonally in both anterograde and retrogradedirections. These data advance our understanding of AAV9 distribution inthe primate brain and provides support for its use in the treatment ofneurological disease with a substantial cortico-striatal pathology suchas Huntington's disease.

As clinical development of neurological gene therapy with vectors basedon adeno-associated virus (AAV) becomes more common, the behavior ofspecific serotypes of AAV in the primate brain is becoming moreimportant. This is particularly true in the context of more efficientand advanced vector infusion technologies that are driving clinicaldevelopment of neurosurgical interventions in diseases such asParkinson's disease (Richardson et al. (2011) Mol Ther 19: 1048-1057)and rare neurological disorders such as aromatic L-amino aciddecarboxylase (AADC) deficiency (San Sebastian et al. (2014) Mol TherMethods Clin Dev 3). This new clinically applied technique employsintra-operative MRI to visualize parenchymal infusions of AAV2. Itsutility is derived from the remarkable correlation between distributionof MRI contrast reagent and eventual transgene expression. However, thisclose correlation breaks down somewhat with AAV9. Expression of GFPextended into a volume significantly (˜3-fold) beyond the volume of theinfusion, emphasizing the important role of interstitial or perivasculartransport processes engaged as result of the initial pressurizedinfusion (CED) (Hadaczek et al. (2006) Mol Ther 14: 69-78). In the caseof AAV2, we would argue that the avidity of the vector for abundantheparan sulphate proteoglycans (Summerford et al. (1998) J Virol 72:1438-1445) helps to restrict AAV2 to the infusion site and matches thedistribution of transgene expression closely to the distribution of MRIcontrast reagent. In contrast, the primary receptor for AAV9 is not HSPG(Shen et al. (2011) J Biol Chem 286:13532-13540) and this vector maythus engage the perivasculature to achieve a much greater volume ofexpression for a given infusion volume.

AAV9 evinces a broad tropism in neural tissue (Gray et al. (2011) MolTher 19:1058-1069; Hinderer et al. (2014) Molecular Therapy—Methods &Clinical Development 1; Foust et al. (2009) Nat Biotechnol 27: 59-65,transducing both neurons and astrocytes as well as perhaps other celltypes. The ability of AAV9 to transduce antigen-presenting cells (APC)in the brain has raised concerns with respect to expression of foreign(non-self) proteins (Ciesielska et al. (2013) Mol Ther 21: 158-166;Samaranch et al. (2014) Mol Ther 22: 329-337; Forsayeth and Bankiewicz(2015) Mol Ther 23: 612) in APC and the consequent engagement ofneurotoxic adaptive immune responses. This, of course, is not likely tobe a problem when self-proteins are expressed, but in the present studywe observed, as previously, the activation of Iba1 and upregulation ofMHC-II on astrocytes and microglia. Both types of glia are brain APCthat have unique individual functions. However, we saw no evidence ofmicroglial transduction by AAV9-GFP even though these cells were clearlyresponsive to GFP presentation. Our conclusion is that astrocytes arethe key APC with respect to adaptive responses to GFP expression.

One of the most striking discoveries about the behavior of AAV serotypesin the brain has been the phenomenon of axonal transport. The ability ofneurons to transport intact AAV particles over long distances was firstdescribed for AAV2, although this same phenomenon has been described forHerpes simplex (Costantini et al. (1999) Hum Gene Ther 10: 2481-2494;Diefenbach et al. (2008) Rev Med Virol 18:35-51; Lilley et al. (2001) JVirol 75: 4343-4356; and McGraw and Friedman (2009) J Virol83:4791-4799) and Rabies virus (Gillet et al. (1986) J Neuropathol ExpNeurol 45:619-634; Kelly and Strick (2000) J Neurosci Methods 103:63-71;Klingen et al. (2008) J Virol 82: 237-245; Larsen et al. (2007) FrontNeural Circuits 1:5). In contrast to the primarily retrograde transportof the above viruses, AAV2 undergoes anterograde transport in CNSneurons; that is, particles of AAV2 are transported intact from neuronalcell bodies to synaptic terminals where they are released to be taken upby neurons in distal locations. See, Ciesielska, et al. (2011) Mol Ther19:922-927; Kells et al. (2012) Neurobiol Dis 48: 228-235; Kells et al.(2009) Proc Natl Acad Sci USA 106:2407-2411. This phenomenon requiresvery efficient distribution and transduction at the primary transductionlocation and it may explain why it was not discovered earlier since onlyCED can really achieve this degree of efficiency. Infusion of AAV2 intoNHP thalamus results in widespread cortical expression of transgene,primarily pyramidal neurons located in cortical lamina V/VI. Similarly,transduction of NHP putamen or rat striatum with AAV2 results intransgene expression in cell bodies within SNr, which receivesprojections from striatal GABAergic neurons, but not SNc, which projectsto striatum. In contrast to the anterograde transport of AAV2, AAV6 istransported in an exclusively retrograde direction and is almost asneurotropic as AAV2 (Salegio et al. (2012) Gene Ther. 20(3):348-52; SanSebastian et al. (2013) Gene Ther 20: 1178-1183).

Axonal transport of AAV9 was found in this study to be bi-directional.Infusion of AAV9-GFP into putamen led to transgene expression incortico-striatal neurons that project to putamen and GFP expression wasalso found in SNc neurons, thereby confirming retrograde transport ofthis serotype. This phenomenon, significantly more efficient than seenwith AAV6 (San Sebastian et al. (2013) Gene Ther 20: 1178-1183) isvaluable in devising therapies for Huntington's disease in whichdegeneration of both basal ganglionic and cortico-striatal neurons iscentral to the neuropathology of the disease (Berardelli et al. (1999)Mov Disord 14:398-403). Efficient transduction of human putamen with atherapeutic AAV9 enables cortical projections to striatum to be targetedas well.

Additionally, however, the vector was transported in an anterogradedirection to SNr and to STN. Labeling of STN neurons was highlydependent on vector dose, more so than in SNr, reflecting therequirement for transport of AAV9-GFP via an indirect route throughglobus pallidus (GP), by axonal and/or perivascular transport. Theability of AAV9 to spread efficiently beyond the initial putaminalinfusion volume suggests a powerful perivascular mechanism.Nevertheless, the phenomenon of bidirectional axonal transport of AAV9may explain in part why this vector is regarded with such enthusiasm forapplications in which very widespread distribution of vector isessential.

Example 5: Delivery of Htt Repressors

Htt repressors (ZFP-TFs, TALE-TFs, CRISPR/Cas-TFs), for example asdescribed in U.S. Publication Nos. 20150056705; 20110082093;20130253040; and 20150335708 and herein are delivered to the striatum ofHD model mice, NHP or human subjects using viral (e.g., AAV such asAAV9) as set forth in Example 1.

The Htt repressors exhibit widespread expression and reduce theformation of Htt expression, Htt aggregates; reduce apoptosis; and/orreduce motor deficits (e.g., clasping) in the subjects and are effectivein the prevention and/or treatment of HD.

Example 6: Repression of Mutant Htt in HD Patient Fibroblasts and inNeurons Derived from HD Patient Stem Cells

ZFP 46025 and 45643 selectively represses mutant HTT in CAG18/45fibroblasts derived from HD patients (FIG. 8). mRNA encoding the GFPcontrol; ZFP 46025 and ZFP 45643 (0.1, 1, 10 or 100 ng per 50,000 cells)were transfected into HD fibroblasts GM02151 (Coriell Cell Repository)using a Nucleofactor (Lonza). Twenty-four hours after transfection, HTTexpression levels were measured by qRT-PCR. Wt Htt (CAG18) and mutantHtt (CAG45) levels in each sample were measured by a customallele-specific qPCR assay based on SNP rs363099 C/T (exon 29) intriplicates, and normalized to the levels of GAPDH. The Htt/GAPDH ratiofor ZFP samples were scaled to that of the mock transfected sample (setto 1). Data are expressed as mean±SD. The data shows selectiverepression of the mutant Htt allele (CAG45) by both ZFP 46025 and ZFP45643.

ZFP 46025 and 45643 selectively repress mutant Htt in CAG21/38fibroblasts derived from HD patients (FIG. 9), mRNA for the GFP control,ZFP 46025 and 45643 (0.1, 1, 10 or 100 ng per 50,000 cells) weretransfected into HD fibroblasts ND30259 (Coriell Cell Repository) usinga Nucleofactor (Lonza). Twenty-four hours after transfection, Httexpression levels were measured by qRT-PCR. Wt Htt (CAG21) and mutantHtt (CAG38) levels in each sample were measured by a customallele-specific qPCR assay based on SNP rs362331 C/T (exon 50) intriplicates, and normalized to the levels of GAPDH. The Htt/GAPDH ratiofor ZFP samples were scaled to that of the mock transfected sample (setto 1). Data are expressed as mean±SD. The data shows selectiverepression of the mutant HTT allele (CAG45) by both ZFP 46025 and 45643.

Data in FIGS. 8 and 9 show that ZFP 46025 and 45643 can selectivelyrepress transcription from mutant Htt alleles in patient-derived cellsthat contain different CAG repeat lengths on both wild type and mutantHtt alleles.

ZFP 46025 and 45643 selectively repress mutant Htt in CAG17/48 neuronsby transient mRNA transfection (FIG. 10). mRNA for the GFP control, ZFP46025 and 45643 (15, 150, 300 or 1,500 ng per 150,000 cells) weretransfected into neurons differentiated from HD embryonic stem cells(ESC) GENEA020 (GENEA/CHDI) using a Nucleofactor (Lonza). Two days aftertransfection, Htt expression levels were measured by qRT-PCR. Wild typeHtt (CAG17) and mutant Htt (CAG48) levels in each sample were measuredby an allele-specific qPCR assay based on SNP rs362307 in exon 67(Applied Biosystems) in triplicates, and normalized to the levels ofGAPDH. The Htt/GAPDH ratio for ZFP samples were scaled to that of themock transfected sample (set to 1). Data are expressed as mean±SD. Thedata shows selective repression of the mutant Htt allele (CAG48) by bothZFP 46025 and 45643 in HD neurons.

The experiments were also carried out on the differentiated CAG17/48neurons where the ZFPs using were delivered using either AAV6 or AAV9viral vectors. AAV6 vectors encoding ZFP 46025, 45643 or GFP controlwere used to infect neurons differentiated from HD embryonic stem cells(ESC) GENEA020 (GENEA/CHDI) in duplicate. AAV doses used were 1E+4,5E+4, or 1E+5 vector genome (vg) per cell for ZFPs and 1E+5 vg per cellfor GFP. Twenty-one days after infection, HTT expression levels weremeasured by qRT-PCR. Wt Htt (CAG17) and mutant Htt (CAG48) levels ineach sample were measured by an allele-specific qPCR assay based on SNPrs362307 in exon 67 (Applied Biosystems) in triplicates, and normalizedto the levels of GAPDH. The Htt/GAPDH ratio for ZFP samples were scaledto that of the mock transfected sample (set to 1). Data are expressed asmean±SD (FIG. 11A).

AAV9 vectors encoding ZFP 46025, 45643 or GFP control were used toinfect neurons differentiated from HD embryonic stem cells (ESC)GENEA020 (GENEA/CHDI) in duplicate. AAV doses used were 1E+5, 5E+5 or5E+6 vector genome (vg) per cell for ZFPs and 5E+6 vg per cell for GFP.Twenty-one days after infection, HTT expression levels were measured byqRT-PCR. Wild type Htt (CAG17) and mutant Htt (CAG48) levels in eachsample were measured by an allele-specific qPCR assay based on SNPrs362307 in exon 67 (Applied Biosystems) in triplicates, and normalizedto the levels of GAPDH. The Htt/GAPDH ratio for ZFP samples were scaledto that of the mock transfected sample (set to 1). Data are expressed asmean±SD (FIG. 11B).

Example 7: ZFP 46025 and 45643 Rescue Cellular Phenotypes that areRelated to HD

Previous studies have shown phenotypic changes associated with expandedCAG repeats in HD patient-derived cells (Jung-il et al., (2012)Biochemical Journal, 446(3), 359-371; HD IPSC Consortium, (2012) CellStem Cell, 11(2), 264-278; An et al., (2012) Cell Stem Cell, 11(2),253-263). In agreement with these published findings, we found thatCAG17/48 neurons had a significant decrease in intracellular ATP levelscompared to non-HD (normal) neurons (FIG. 12A). Twenty-one days afterneurons were infected with lentiviral vectors encoding ZFP 46025 or ZFP45643, intracellular ATP levels increased 1.7- and 1.8-fold compared tocontrol cells, respectively, indicating that mutant Htt silencingrescues the energetic defect in HD neurons. Another phenotype of HDneurons in vitro is the increased susceptibility to programmed celldeath. With growth factor withdrawal, the percentage of CAG17/48 neuronsundergoing apoptosis was 4-5-fold higher than that of normal neurons(FIG. 12B). At 12 days after lentiviral infection, followed by two daysof growth factor withdraw, ZFP 46025 and 45643 reduced the number ofapoptotic cells to that seen in wild-type cells.

Intracellular ATP levels of cultured neurons, derived from an HD patient(CAG17/48) or a normal subject, were measured using the CellTiter-Glo®Luminescent Assay (Promega), where cell numbers in each sample weredetermined using the ApoLive-Glo® assay (Promega). Neurons were infectedin triplicate with LV expressing either YFP-Venus or ZFP-TF (45643 or46025-KOX-2A-Venus) at an MOI of 500.

At 21 days after lentiviral infection, intracellular [ATP] levels inneurons were measured using the CellTiter-Glo Luminescent Assay(Promega) according to manufacturer's instructions. Luminescence valueswere normalized to the cell number in each sample. ATP level per cellvalues from different cells/treatment were then normalized to that ofmock-infected HD neurons (set as 1). Data (FIG. 12A) are expressed asmean±SD.

Cell death of HD and non-HD neurons induced by growth factor withdrawalwas measured using a terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) assay. Neurons were infected in triplicate with LVexpressing either YFP-Venus or ZFP-TF (45643 or 46025-KOX-2A-Venus) atan MOI of 500. The cells were cultured for 12 days then media waschanged to fresh neurobasal media without any additive (growth factors).Cells were kept in this growth factor withdrawal media for 48 hours.TUNEL assay was performed using the ApoBrdU Red DNA fragmentation kit(BioVision). Neurons were fixed with 4% paraformaldehyde on ice for 15min. Apoptosis was assessed by quantifying TUNEL-positive cellsaccording to the manufacturer recommendations (ApoBrdU Red DNAfragmentation kit, BioVision). Flow cytometry was used to measureapoptosis by anti-BrdU-Red staining. Data (FIG. 12B) are expressed asmean±SD.

Thus, repressors as described herein provide therapeutic benefits byrescuing HD-related phenotypes, including, but not limited to,decreasing cell death, increasing cell function (as measured byintracellular ATP levels) and decreasing cell susceptibility toapoptosis as compared to untreated cells.

Example 8: ZFP 46025 and ZFP 45643 Repress Mutant Htt in Mouse Striatum

In vivo activity of ZFPs were tested in HdhQ50/Hdh+ (Q50) heterozygousmice (White et al. (1997) Nature Genetics 17: 404-410) by intrastriatalinjection of AAV9 vectors encoding ZFP 46025, ZFP 45643 or GFP control.The Q50 mice contain a knock-in allele where exon 1 of the endogenousmouse Hdh gene was replaced with exon 1 of the human Htt gene with 48CAGs. At 5 weeks after injection, allele-specific qRT-PCR analysis oftreated striatum showed that ZFP 45643 and ZFP 46025 repressed themutant Htt allele (Q50) by 79% and 74%, respectively, relative tovehicle injected control; the wild type allele (Q7) was not regulated byeither ZFP (FIGS. 13A and 13B). Activity of ZFP 45643 was also tested at12 weeks after injection (FIG. 13C), and significant repression (70%) ofmutant Htt (Q50) was observed with no repression of the wild type allele(Q7). No overt toxicity was observed in any of the animals over thecourse of the study. Behavioral studies (e.g., clasping studies) arealso performed and show that the repressors as described herein providea therapeutic (clinical) benefit in vivo.

HdhQ50/Hdh+ (Q50) heterozygous mice (mixed gender) received bilateralintrastriatal injection of AAV9 vectors (ZFP 46025, 45643 or GFP, n=4per group) or formulation buffer (vehicle, n=3) at 10-11 weeks of age.Two 3-μl injections were placed into each striatum (for a total of 6 μland 1.1E+10 vector genome per striatum). The coordinates used for theanterior infusion was A/P+1.4, M/L+/−1.7, D/V−3.5 and for the posteriorinfusion was A/P+0.2, M/L+/−2.3, D/V−3.2. At five weeks (ZFP 46025 and45643) and 12 weeks (ZFP 45643) after injection, mice were sacrificedand each striatum was dissected into three (anterior, middle andposterior) slices for RNA isolation and qRT-PCR analysis. Expressionfrom the mutant Htt allele (Q50) and the wt allele (Q7) were measured byallele-specific qPCR assays, Htt levels were normalized to the geometricmean of ATP5B, RPL38 and EIF4A2 levels. (ns: p>0.05, *: p<0.05, **:p<0.01, ***: p<0.001, ****:p<0.0001, one way ANOVA with Sidak test).

As shown in FIG. 13, allele-specific qRT-PCR analysis of treatedstriatum showed that ZFP 45643 and ZFP 46025 repressed the mutant Httallele (Q50) by 79% and 74%, respectively, relative to vehicle injectedcontrol; the wild type allele (Q7) was not regulated by either ZFP.Activity of ZFP 45643 was also tested at 12 weeks after injection andsignificant repression (70%) of mutant Htt (Q50) was observed with norepression of the wild type allele (Q7).

Thus, Htt repressors as described herein exhibit widespread expressionand reduce the formation of Htt expression, Htt aggregates; reduceapoptosis; and/or reduce motor deficits (e.g., clasping) in subjects andare effective in the prevention and/or treatment of HD.

Example 9: Measuring Therapeutic Efficacy of ZFP 46025 and ZFP 45643

HD patients are treated with ZFP 46025 or ZFP 45643 at varying doses. HDpatients are evaluated using the UHDRS scale and show improvementfollowing treatment. Efficacy is also measured by PET imaginganalysisusing ¹⁸FMNI-659, a PET tracer for PDE10A, and MRI. In brief, regions ofthe brain (e.g. areas within putamen) that are exposed to ZFP (e.g. AAV9vector encoding the ZFP) are identified by Gadolinium contrast agentthat is mixed with the ZFP formulation and Mill; before and aftertreatment, patients are given approximately 5 mCi of ¹⁸FMNI-659 at amass dose of about 5 μg over a three minute infusion period. Serial 3DPET images are acquired for a period of 90 minutes using a PET scanner.MM images are also obtained using a Mill scanner, and the PET images arealigned with the Mill images to generate anatomy-based visuals foranalysis. Standard uptake values are calculated for the basal ganglianuclei (which includes the globus pallidus, caudate, and putamen(striatum)) and normalized to a reference region such as the cerebellum(Russell et al, ibid). PDE10A PET signals in brain regions that areexposed to the ZFP, identified by MM at time of treatment, are measuredafter treatment and compared to signal levels in the same region beforetreatment. Treatment of the HD patients with ZFP46025 or ZFP45643protects the patients from any additional loss of medium spiny neuron(measured by PDE10A levels) and from the further development of overtclinical symptoms. In some patients, treatment with the ZFPs reversesthe symptoms of HD.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference for all purposes in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A non-naturally occurring zinc finger proteinthat binds to an Htt gene, the zinc finger protein comprising 5 zincfinger domains ordered F1 to F5, wherein the zinc finger domainscomprise the recognition helix regions sequences shown in a single rowof Table
 1. 2. A fusion protein comprising a zinc finger protein ofclaim 1 and a functional domain.
 3. The fusion protein of claim 2,wherein the functional domain a transcriptional activation domain, atranscriptional repression domain, and a nuclease domain.
 4. Apolynucleotide encoding one or more zinc finger proteins of claim
 1. 5.An AAV vector comprising the polynucleotide of claim
 4. 6. A host cellcomprising one or more zinc finger proteins of claim
 1. 7. Apharmaceutical composition comprising one or more polynucleotidesaccording to claim
 4. 8. A pharmaceutical composition comprising one ormore AAV vectors according to claim
 5. 9. A method of modifyingexpression of an Htt gene in a cell, the method comprising administeringto the cell one or more polynucleotides according to claim
 4. 10. Themethod of claim 9, wherein the Htt gene comprises at least wild-typeand/or one mutant allele.
 11. The method of any claim 9, wherein thefusion protein comprises a repression domain and expression of the Httgene is repressed.
 12. The method of claim 9, wherein the cell is aneuronal cell.
 13. The method of claim 12, wherein the neuronal cell isin a brain.
 14. The method of claim 13, wherein the neuronal cell is inthe striatum of the brain.
 15. A method of treating and/or preventingHuntington's Disease in a subject in need thereof, the method comprisingadministering one or more polynucleotides according to claim 5 to thesubject in need thereof.